Dna methylation diagnostic test for breast cancer

ABSTRACT

The present subject matter provides, inter alia, a method of diagnosing invasive ductal cancer or detecting invasive ductal cancer risk in a subject who has ductal carcinoma in situ, a method of treating breast cancer in a subject detected to have invasive ductal cancer or determined to be at risk of invasive ductal cancer, compositions for detecting invasive presence or potential in ductal carcinoma in situ in a subject, and kits including reagents and composition for detecting invasive presence or potential in ductal carcinoma in situ in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/611,910, filed Dec. 29, 2017, which is herebyincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Invasive breast cancer represents a quarter of all cancers diagnosedworldwide and its rate increases by 0.3% every year. Ductal carcinoma insitu (DCIS) is considered to be a pre-invasive form of breast cancer.Almost a quarter of all new breast cancers diagnosed in the UnitedStates are DCIS. However, patients with DCIS are treated with surgeryand radiation, identically to patients presenting with invasive disease.Not all women with untreated DCIS will develop invasive ductal cancer(IDC). At present, there is no tool to predict concurrent or subsequentinvasion in DCIS. This results in overtreatment of a significant numberof patients who carry the stigma of cancer.

BRIEF SUMMARY OF THE INVENTION

The present subject matter provides, inter alia, methods, compounds,compositions, kits, and systems for diagnosing IDC or detecting IDC riskin a subject who has DCIS, treating breast cancer in a subject detectedto have IDS or determined to be at risk of IDC, and detecting invasivecompetent DCIS and invasive incompetent DCIS in a subject.

In an aspect, provided herein is a method for detecting the presence,absence, and/or level of methylation at one or more sites set forth inTable 1. In embodiments, the level of methylation at a site set forth inTable 1 is detected.

In an aspect, included herein is method of detecting methylation orunmethylation of a DCIS cell DNA molecule of a subject. In embodiments,the method comprises (i) contacting an isolated DCIS cancer cellproliferation DNA molecule from the subject with a bisulfite saltthereby forming a reacted DCIS cancer cell proliferation DNA molecule;and (ii) detecting the presence or absence of uracil in the reacted DCIScancer cell proliferation DNA molecule at a methylation site set forthin Table 1, thereby detecting methylation or unmethylation of the DCIScancer cell proliferation DNA molecule of the subject.

In embodiments, provided herein is a method of detecting methylation orunmethylation of a DCIS cancer cell proliferation DNA molecule of asubject. In embodiments, the method comprises (i) isolating a DCIScancer cell proliferation DNA molecule from a DCIS cancer cellproliferation of the subject thereby forming an isolated DCIS cancercell proliferation DNA molecule; (ii) contacting the isolated DCIScancer cell proliferation DNA molecule with a bisulfite salt therebyforming a reacted DCIS cancer cell proliferation DNA molecule; and (iii)detecting the presence or absence of uracil in the reacted DCIS cancercell proliferation DNA molecule at a methylation site set forth in Table1, thereby detecting methylation or unmethylation of the DCIS cancercell proliferation DNA molecule of the subject.

Also provided herein is a method of detecting methylation orunmethylation of a plurality of DCIS cancer cell proliferation DNAmolecules of a subject. In embodiments, the method comprises (i)contacting a plurality of isolated DCIS cancer cell proliferation DNAmolecules from the subject with a bisulfite salt thereby forming aplurality of reacted DCIS cancer cell proliferation DNA molecules; and(ii) detecting the level of uracil in the plurality of reacted DCIScancer cell proliferation DNA molecules at a plurality of methylationsites set forth in Table 1, thereby detecting the level of methylationor unmethylation in the plurality of DCIS cancer cell proliferation DNAmolecules of the subject.

Embodiments also provide a method of detecting methylation orunmethylation of a plurality of DCIS cancer cell proliferation DNAmolecules of a subject. In embodiments, the method comprises (i)isolating a plurality of DCIS cancer cell proliferation DNA moleculesfrom the DCIS cancer cell proliferation of the subject thereby forming aplurality of isolated DCIS cancer cell proliferation DNA molecules; (ii)contacting the plurality of isolated DCIS cancer cell proliferation DNAmolecules with a bisulfite salt thereby forming a plurality of reactedDCIS cancer cell proliferation DNA molecules; and detecting the level ofuracil in the plurality of reacted DCIS cancer cell proliferation DNAmolecules at a plurality of methylation sites set forth in Table 1,thereby detecting the level of methylation or unmethylation in theplurality of DCIS cancer cell proliferation DNA molecules of thesubject.

In an aspect, further provided is a method of detecting a risk ofdeveloping IDC in a subject who has DCIS. In embodiments, the methodcomprises (i) contacting an isolated DCIS cancer cell proliferation DNAmolecule from the subject with a bisulfite salt thereby forming areacted DCIS cancer cell proliferation DNA molecule; and (ii) detectingthe presence or absence of uracil in the reacted DCIS cancer cellproliferation DNA molecule at a methylation site set forth in Table 1,thereby detecting the risk for developing IDC in the subject. Inembodiments, the method comprises (i) contacting a plurality of isolatedDCIS cancer cell proliferation DNA molecules from the subject with abisulfite salt thereby forming a plurality of reacted DCIS cancer cellproliferation DNA molecules; and (ii) detecting the level of reactedDCIS cancer cell proliferation DNA molecules in the plurality of reactedDCIS cancer cell proliferation DNA molecules having a uracil at aplurality of methylation sites set forth in Table 1, thereby detectingthe risk for IDC in the subject.

Also provided is a method of detecting IDC or a risk of developing IDCin a subject. In embodiments, the method includes (i) isolating a DCIScancer cell proliferation DNA molecule from DCIS tissue of the subjectthereby forming an isolated DCIS cancer cell proliferation DNA molecule;(ii) contacting the isolated DCIS cancer cell proliferation DNA moleculewith a bisulfite salt thereby forming a reacted DCIS cancer cellproliferation DNA molecule; and (iii) detecting the presence or absenceof uracil in the reacted DCIS cancer cell proliferation DNA molecule ata methylation site set forth in Table 1, thereby detecting the risk fordeveloping IDC in the subject. In embodiments, the method comprises (i)isolating a plurality of DCIS cancer cell proliferation deoxyribonucleicacid (DNA) molecules from DCIS tissue of the subject thereby forming aplurality of isolated DCIS cancer cell proliferation DNA molecules; (ii)contacting the plurality of isolated DCIS cancer cell proliferation DNAmolecules with a bisulfite salt thereby forming a plurality of reactedDCIS cancer cell proliferation DNA molecules; and (iii) detecting thelevel of reacted DCIS cancer cell proliferation DNA molecules in theplurality of reacted DCIS cancer cell proliferation DNA molecules havinga uracil at a plurality of methylation sites set forth in Table 1,thereby detecting the risk for IDC in the subject.

In embodiments, the detection of the presence, absence, or level ofuracil in a reacted DCIS cancer cell proliferation DNA molecule at amethylation site set forth in Table 1 comprises amplifying the reactedDCIS cancer cell proliferation DNA molecule [e.g., by a polymerase chainreaction (PCR)] to produce an amplicon, and determining whether athymidine is present at the nucleotide position of the amplicon thatcorresponds to the nucleotide position of the uracil in the reacted DCIScancer cell proliferation DNA molecule.

Aspects also include a method of treating or preventing IDC a subjectdetected to have a risk of IDC or diagnosed as having IDC. Inembodiments, the method includes administering to the subject atreatment to treat or prevent IDC or directing the subject to obtaintreatment to treat or prevent IDC.

Also included herein is a DNA molecule at least 5 to 100 nucleotides inlength comprising a uracil-containing sequence that is identical to asequence of at least 5 contiguous nucleotides within a sequence chosenfrom SEQ ID NO:1 to SEQ ID NO:242.

In embodiments, provided herein is an oligonucleotide comprising auracil-containing sequence that is identical to a sequence of at least5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100,100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180,180-190, or 190-200 contiguous nucleotides within the sequence chosenfrom SEQ ID NO:1 to SEQ ID NO:242, or an oligonucleotide that is anamplicon or is identical to an amplicon of a methylation site-containingsequence of at least 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150,150-160, 160-170, 170-180, 180-190, or 190-200 contiguous nucleotideswithin the sequence chosen from SEQ ID NO:1 to SEQ ID NO:242.

Aspects of the present subject matter also include a deoxyribonucleicacid chosen from SEQ ID NO:243 to SEQ ID NO: 356, wherein the nucleicacid is hybridized to a complementary DNA sequence comprising uridine orcytosine.

Also provided is a kit comprising a plurality of nucleic acids eachindependently comprising SEQ ID NO: 242 to SEQ ID NO: 356, wherein eachnucleic acid of the plurality is unique.

In embodiments, included herein is a system for detecting methylation orunmethylation of a DCIS cancer cell proliferation DNA molecule of asubject. In embodiments, the system provides at least one processor; andat least one memory including program code which when executed by the atleast one processor provides operations comprising: contacting anisolated DCIS cancer cell proliferation DNA molecule from the subjectwith a bisulfate salt thereby forming a reacted DCIS cancer cellproliferation DNA molecule; detecting the presence or absence of uracilin the reacted DCIS cancer cell proliferation DNA molecule at amethylation site set forth in Table 1, thereby detecting methylation orunmethylation of the DCIS cancer cell proliferation DNA molecule of thesubject; generating a diagnosis for the subject based at least in parton the presence or absence of uracil in the reacted DCIS cancer cellproliferation DNA molecule at the methylation site set forth in Table 1;and providing, via a user interface, the diagnosis or prognosis for thesubject. In embodiments, the system provides at least one processor; andat least one memory including program code which when executed by the atleast one processor provides operations comprising: contacting theplurality of isolated DCIS cancer cell proliferation DNA molecules witha bisulfite salt thereby forming a plurality of reacted DCIS cancer cellproliferation DNA molecules; detecting the level of reacted DCIS cancercell proliferation DNA molecules in the plurality of reacted DCIS cancercell proliferation DNA molecules having a uracil at a methylation siteset forth in Table 1 thereby detecting the level of methylation orunmethylation in the plurality of DCIS cancer cell proliferation DNAmolecules of the subject; generating a diagnosis for the subject basedat least in part on the level of methylation or unmethylation at theplurality of methylation sites set forth in Table 1; and providing, viaa user interface, the diagnosis or prognosis for the subject.

Aspects also include a system for detecting methylation or unmethylationof a DCIS cancer cell proliferation DNA molecule of a subject. Inembodiments, the system comprises at least one processor and at leastone memory including program code which when executed by the at leastone processor provides operations. In embodiments, the operationscomprise (i) isolating a DCIS cancer cell proliferation DNA moleculefrom a DCIS cancer cell proliferation of the subject thereby forming anisolated DCIS cancer cell proliferation DNA molecule; (ii) contactingthe isolated DCIS cancer cell proliferation DNA molecule with abisulfite salt thereby forming a reacted DCIS cancer cell proliferationDNA molecule; (iii) detecting the presence or absence of uracil in thereacted DCIS cancer cell proliferation DNA molecule at a methylationsite set forth in Table 1, thereby detecting methylation orunmethylation of the DCIS cancer cell proliferation DNA molecule of thesubject; (iv) generating a diagnosis for the subject based at least inpart on the presence or absence of uracil in the reacted DCIS cancercell proliferation DNA molecule at the methylation site set forth inTable 1; and (v) providing, via a user interface, the diagnosis orprognosis for the subject.

Also provided herein is a system for detecting methylation orunmethylation of a plurality of DCIS cancer cell proliferation DNAmolecules of a subject. In embodiments, the system comprises at leastone processor and at least one memory including program code which whenexecuted by the at least one processor provides operations. Inembodiments, the operations comprise (i) isolating a plurality of DCIScancer cell proliferation DNA molecules from the DCIS cancer cellproliferation of the subject thereby forming a plurality of isolatedDCIS cancer cell proliferation DNA molecules; (ii) contacting theplurality of isolated DCIS cancer cell proliferation DNA molecules witha bisulfite salt thereby forming a plurality of reacted DCIS cancer cellproliferation DNA molecules; (iii) detecting the level of reacted DCIScancer cell proliferation DNA molecules in the plurality of reacted DCIScancer cell proliferation DNA molecules having a uracil at a methylationsite set forth in Table 1 thereby detecting the level of methylation orunmethylation in the plurality of DCIS cancer cell proliferation DNAmolecules of the subject; (iv) generating a diagnosis for the subjectbased at least in part on the level of methylation or unmethylation atthe plurality of methylation sites set forth in Table 1; and (v)providing, via a user interface, the diagnosis or prognosis for thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a set of images, FIG. 1C is a table, and FIGS. 1B and 1D areheatmap images relating to the epigenetic analysis of DCIS. FIG. 1A:Extraction of cancer cells from DCIS containing ducts. DCIS slide beforeand after a laser cut. FIG. 1B: DNA methylation patterns at promoters inDCIS. Heatmap was done for promoter regions with available DNAmethylation information (20,910 regions). Green represents low level ofDNA methylation, red indicates a high level of DNA methylation. FIG. 1C:Pathology data for the samples included in the study. FIG. 1D: DNAmethylation status of 140 cytosines separates “invasion incompetent” and“invasion competent” DCIS. Green represents low level of DNAmethylation, red indicates a high level of DNA methylation. “ER” means“estrogen receptor” and “PR” means “progesterone receptor.” (+) meanspositive for estrogen and progesterone receptor staining. (−) meansnegative for estrogen and progesterone receptor staining. DCIS_IDC_11,DCIS_IDC_13(+), and DCIS_IDC_14(+) are samples from patients with DCISwith adjacent IDC. DCIS_3(−) and DCIS_6(−), INV, are samples frompatients without IDC with DCIS negative for estrogen and progesteronereceptor staining. DCIS_4, DCIS_5(+), DCIS_7(+), DCIS_8(+), andDCIS_1(+) are samples from patients without IDC with DCIS positive forestrogen and progesterone receptor staining (with the exception thatstatus of estrogen receptor and progesterone receptor staining is notknown for the DCIS_4).

FIG. 2 depicts a block diagram illustrating an exemplary breast cancerdiagnostics system.

FIG. 3 depicts a flowchart illustrating an exemplary process fordiagnosing breast cancer.

FIG. 4 depicts a flowchart illustrating an exemplary process fordiagnosing breast cancer.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are, inter alia, compositions, methods, and kits fordetecting methylated and/or unmethylated DNA. In some aspects, thepresent disclosure includes compositions, methods, and kits fordetecting methylated and/or unmethylated DNA from ductal carcinoma insitu.

The following definitions are included for the purpose of understandingthe present subject matter and for constructing the appended patentclaims. Abbreviations used herein have their conventional meaning withinthe chemical and biological arts.

Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in oncology, cellculture, molecular genetics, epigenetics, and biochemistry).

As used herein, the term “about” in the context of a numerical value orrange means ±10% of the numerical value or range recited or claimed,unless the context requires a more limited range.

In the descriptions above and in the claims, phrases such as “at leastone of” or “one or more of” may occur followed by a conjunctive list ofelements or features. The term “and/or” may also occur in a list of twoor more elements or features. Unless otherwise implicitly or explicitlycontradicted by the context in which it is used, such a phrase isintended to mean any of the listed elements or features individually orany of the recited elements or features in combination with any of theother recited elements or features. For example, the phrases “at leastone of A and B;” “one or more of A and B;” and “A and/or B” are eachintended to mean “A alone, B alone, or A and B together.” A similarinterpretation is also intended for lists including three or more items.For example, the phrases “at least one of A, B, and C;” “one or more ofA, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, Balone, C alone, A and B together, A and C together, B and C together, orA and B and C together.” In addition, use of the term “based on,” aboveand in the claims is intended to mean, “based at least in part on,” suchthat an unrecited feature or element is also permissible.

It is understood that where a parameter range is provided, all integerswithin that range, and tenths thereof, are also provided by theinvention. For example, “0.2-5 mg” discloses 0.2 mg, 0.3 mg, 0.4 mg, 0.5mg, 0.6 mg etc. up to and including 5.0 mg. Additionally, where twovalues for a parameter are disclosed, then a range of all values betweenand including those two values is also disclosed. For example, “1, 2,and 3” discloses, e.g., 1-2, 1-3, and 2-3.

A small molecule is a compound that is less than 2000 daltons in mass.The molecular mass of the small molecule is preferably less than 1000daltons, more preferably less than 600 daltons, e.g., the compound isless than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100daltons.

The term “ductal carcinoma in situ cancer cell proliferation” (alsorecited as “DCIS cancer cell proliferation” herein) refers to abiological sample comprising one or more cells obtained or provided fromDCIS tissue of a subject. In embodiments, the DCIS cancer cellproliferation comprises a biopsy taken, e.g., from an abnormality [suchas mammographic or magnetic resonance imaging (MRI) abnormality] in thebreast of a subject. Non-limiting examples of mammographic abnormalitiesmay include or be referred to as suspicious calcifications,microcalcifications, clustered microcalcifications, pleomorphiccalcifications, pleomorphic branching calcifications, or a cluster ofheterogeneous calcifications. In embodiments, a mammographic abnormalityis observed as a mass. In embodiments, the mammographic abnormality isother than a mass (e.g., is a proliferation of cells within or along aduct in a string, sheet, plurality, or population of cells).Non-limiting examples of breast MRI abnormalities may include or bereferred to as an abnormal enhancement, a mass-like enhancement, anon-mass enhancement, a linear non-mass enhancement, an enhancingintraductal mass, or a suspicious enhancing lesion. In embodiments, anMRI abnormality or enhancement is observed as a mass. In embodiments,during a breast MRI, mammogram, ultrasound, or physical exam, a DCIScancer cell proliferation presents as a string, sheet, plurality, orpopulation of cells, a mass, an irregular mass, or a mass lesion, or byultrasound as a hypoechoic mass. In embodiments, the DCIS cancer cellproliferation is substantially free of normal cells. For example, atleast about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, ormore of the cells in the DCIS cancer cell proliferation are dysplasticcells such as DCIS cells. In embodiments, at least 75% of the cells inthe DCIS cancer cell proliferation are DCIS cancer cells.

The term “disease” refers to any deviation from the normal health of amammal and includes a state when disease symptoms are present, as wellas conditions in which a deviation (e.g., infection, gene mutation,genetic defect, etc.) has occurred, but symptoms are not yet manifested.According to the present disclosure, the methods disclosed herein aresuitable for use in a patient that is a member of the Vertebrate class,Mammalia, including, without limitation, primates, livestock anddomestic pets (e.g., a companion animal). Typically, a patient will be ahuman patient.

The terms “subject,” “patient,” “individual,” and the like as usedherein are not intended to be limiting and can be generallyinterchanged. That is, an individual described as a “patient” does notnecessarily have a given disease, but may be merely seeking medicaladvice.

The term “subject” as used herein includes any mammal. In embodiments,the subject is a primate such as a human. In embodiments, the subject isa female. In embodiments, the subject is a male. Non-limiting examplesof mammals include rodents (e.g., mice and rats), elephants, sloths,armadillos, primates (such as lemurs, bushbabies, monkeys, apes, andhumans), rabbits, dogs (e.g., pets or work dogs such as police dogs,military dogs, race dogs, or show dogs), horses (such as race horses andwork horses), donkeys, zebras, tapirs, rhinoceroses, cats (e.g.,domesticated cats and large cats such as lions, cheetahs, tigers, andleopards), whales, dolphins, porpoises, pigs, cattle, and deer.

It must be noted that as used herein and in the appended embodiments,the singular forms “a,” “an,” and “the” include the plural referenceunless the context clearly dictates otherwise. Thus, for example, areference to “a disease,” “a disease state”, “a nucleic acid” or “a CpGsite” is a reference to one or more such embodiments, and includesequivalents thereof known to those skilled in the art and so forth.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a patient suspected or atrisk of having breast cancer and compared to samples from a known breastcancer patient, or a known normal (non-disease) individual. A controlcan also represent an average value gathered from a population ofsimilar individuals, e.g., breast cancer patients or healthy individualswith a similar medical background, same age, weight, etc. A controlvalue can also be obtained from the same individual, e.g., from anearlier-obtained sample, prior to disease, or prior to treatment. One ofskill will recognize that controls can be designed for assessment of anynumber of parameters.

One of skill in the art will understand which controls are valuable in agiven situation and be able to analyze data based on comparisons tocontrol values. Controls are also valuable for determining thesignificance of data. For example, if values for a given parameter arewidely variant in controls, variation in test samples will not beconsidered as significant.

As used herein, the term “diagnosing” and the like includes detecting adisease, disorder, and/or a symptom or feature (e.g., invasiveness)thereof. Diagnosing also includes determining the stage or degree of adisease or disorder. The term “prognosis” refers to a relativeprobability that a certain future outcome may occur in the subject. Forexample, in the context of the present disclosure, prognosis can referto the likelihood that an individual will develop a disease, or thelikely severity of the disease (e.g., severity of symptoms, rate offunctional decline, survival, invasiveness, etc.). The terms are notintended to be absolute, as will be appreciated by any one of skill inthe field of medical diagnostics.

“Treating” or “treatment” of a condition as used herein includespreventing or alleviating a condition, slowing the onset or rate ofdevelopment of a condition, reducing the risk of developing a condition,preventing or delaying the development of symptoms associated with acondition, reducing or ending symptoms associated with a condition,generating a complete or partial regression of a condition, curing acondition, or some combination thereof. With regard to cancer,“treating” or “treatment” may refer to inhibiting or slowing neoplasticor malignant cell growth, proliferation, invasion, or metastasis,preventing or delaying the development of neoplastic or malignant cellgrowth, proliferation, invasion, or metastasis, or some combinationthereof. With regard to a tumor, “treating” or “treatment” includeseradicating all or part of a tumor, inhibiting or slowing tumor growth,invasion, and metastasis, preventing or delaying the development of atumor, or some combination thereof.

As used herein, a “symptom” associated with a disorder includes anyclinical or laboratory manifestation associated with the disorder, andis not limited to what the subject can feel or observe.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10,12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100nucleotides in length. Nucleic acids, including ribonucleic acids (RNA)and deoxyribonucleic acids (DNA), and polynucleotides are a polymers ofany length, including longer lengths, e.g., 200, 300, 500, 1000, 2000,3000, 5000, 7000, 10,000, etc. A nucleic acid of the present inventionwill generally contain phosphodiester bonds, although in some cases,nucleic acid analogs are included that may have alternate backbones,comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate,or O-methylphosphoramidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press); and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made.

The term “bp” and the like refer, in the usual and customary sense, tothe indicated number of base pairs.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids (e.g., genomic sequences or subsequences or codingsequences) or polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same (i.e., 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more identity over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Suchsequences are then said to be “substantially identical.” This definitionalso refers to the compliment of a test sequence. Optionally, theidentity exists over a region that is at least about 10 to about 100,about 20 to about 75, about 30 to about 50 amino acids or nucleotides inlength.

An example of algorithms suitable for determining percent sequenceidentity and sequence similarity are the BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402(1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990),respectively. As will be appreciated by one of skill in the art, thesoftware for performing BLAST analyses is publicly available through thewebsite of the National Center for Biotechnology Information.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Any method known in the art for conjugating an antibodyto the label may be employed, e.g., using methods described inHermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

The term “associated” or “associated with” in the context of a substance(e.g., level of uracil or methylation level in a breast lump or DCIScancer cell proliferation) does not necessarily mean that the disease iscaused by (in whole or in part), or a symptom of the disease is causedby (in whole or in part) the substance or substance activity or function(i.e., level of uracil in the regions of chromosomes assayed).

The term “unmethylated DNA” or “demethylated DNA” means DNA that lacks amethyl group conjugated to cytosine in a segment of the DNA. DNAmethylation typically occurs in a CpG dinucleotide context. In thecontext of the present disclosure, the DNA can be equivalent to a short(2-50, 5-50, 2-300, 2-350 nucleotides, e.g. 5-350 nucleotides) doublestranded or single stranded nucleic acid, a nucleic acid fragment clonedin a plasmid DNA, a nucleic acid fragment amplified from a sample of asubject, and/or synthetically prepared a nucleic acid fragment. DNAmethylation at the 5′ position of cytosine may have the specific effecton gene expression in vivo. DNA methylation may also form the basis ofepigenetic structure, which typically enables a single cell to grow intomultiple organs or perform multiple functions.

The CpG sites or CG sites are regions of DNA where a cytosine nucleotideoccurs next to a guanine nucleotide in the linear sequence of basesalong its length. “CpG” is shorthand for “—C-phosphate-G-”, that is,cytosine and guanine separated by only one phosphate; phosphate linksany two nucleosides together in DNA. The “CpG” notation is used todistinguish this linear sequence from the CG base-pairing of cytosineand guanine. The CpG notation can also be interpreted as the cytosinebeing 5′ prime to the guanine base.

In embodiments, methylation is detected based on a chemical reaction ofsodium bisulfite with DNA that converts unmethylated cytosines of CpGdinucleotides to uracil or UpG. However, methylated cytosine is notconverted in this process, the methods of the present disclosure allowdetermination of methylation status as methylated or unmethylated.

As used herein, an “isolated” or “purified” nucleic acid molecule,polynucleotide, polypeptide, or protein, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or chemical precursors or other chemicals when chemicallysynthesized. Purified compounds are at least 60% by weight (dry weight)the compound of interest. Preferably, the preparation is at least 75%,more preferably at least 90%, and most preferably at least 99%, byweight the compound of interest. For example, a purified compound is onethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w)of the desired compound by weight. Purity is measured by any appropriatestandard method, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis. A purified or isolated polynucleotide [ribonucleic acid (RNA)or deoxyribonucleic acid (DNA)] is free of the genes or sequences thatflank it in its naturally-occurring state. Purified also defines adegree of sterility that is safe for administration to a human subject,e.g., lacking infectious or toxic agents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

Exemplary Epigenetic Signatures

DCIS is the most common type of non-invasive breast cancer. Ductal meansthat the cancer starts inside the milk ducts (which are the “pipes” thatcarry milk from the milk-producing lobules to the nipple), carcinomarefers to any cancer arising in the epithelial tissue of the skin or ofthe lining of the internal organs (including breast tissue), and in situmeans “in its original place.” DCIS is called “non-invasive” because ithasn't spread beyond the milk duct into any normal surrounding breasttissue. However, as disclosed herein, DCIS may be predisposed or proneto becoming invasive. DCIS is frequently found to be associated withinvasive cancer where the DCIS remains localized in the ducts while theadjacent invasive cancer invades out of the ducts. IDC, which stands forinfiltrating ductal carcinoma or invasive ductal carcinoma, is the mostcommon type of invasive breast cancer. Invasive means that the cancer“invades” or spreads to surrounding tissues (e.g., surrounding breasttissues). All together, “invasive ductal carcinoma” refers to cancerthat has broken through the wall of the milk duct and invaded thetissues of the breast. Over time, invasive ductal carcinoma can spreadto the lymph nodes and possibly to other areas of the body.

Provided herein are methods, compounds, compositions, kits, and systemsfor detecting invasive competent DCIS and invasive incompetent DCIS in asubject who has DCIS, treating breast cancer in a subject detected tohave invasive competent DCIS or invasive incompetent DCIS, monitoring asubject who has DCIS for invasive competent DCIS, and distinguishinginvasive competent DCIS from invasive incompetent DCIS in a subject.

In an aspect, included herein are molecular epigenetic diagnosticmethods that reduce breast cancer overdiagnosis and overtreatment. Inembodiments, the treatment of a subject differs based on whether thesubject is determined to have IDC or a risk for IDC. For example, asubject determined to have IDC or a risk of IDC may receive surgery suchas a mastectomy, whereas a subject determined to not have IDC or no riskof IDC may receive less invasive surgery, radiation therapy, hormonaltherapy, or be monitored periodically (e.g., retested for IDC, or IDCrisk according to a method disclosed herein at a future time point, suchas 0.5-5 years from the initial test) without receiving surgery,chemotherapy, radiation therapy, or hormonal therapy.

In embodiments, DCIS can be classified into two groups. One groupcomprises DCIS from patients with potential or concomitant invasion andcharacterized by cancer specific DNA methylation changes (“invasioncompetent DCIS” or “IC-DCIS”), the second group with DCIS is without anyinvasive disease (“invasion incompetent DCIS” or “II-DCIS”) andassociated with DNA methylation pattern more similar to normal cells.IC-DCIS has a risk of becoming invasive in the future. In embodiments, asubject with IC-DCIS may have a substantial risk (e.g., at least a 10%,20%, 30%, 40%, or 50% chance) of having IDC (e.g., within about 1, 2, 3,4, 5, or 10 years).

Not to be bound by theory, it is hypothesized that there are twodifferent epigenetic programs driving DCIS progression, and patientswith the “invasion incompetent” signature are not at risk of developinginvasive ductal cancer over time while patients with DCIS from thesecond epigenetic group having “invasion competent” signature will havea very high chance to develop invasive ductal cancer.

In embodiments, provided herein are epigenetic biomarkers based on DNAmethylation that detect DCIS invasive state or potential and/or predictDCIS progression to an invasive state. In embodiments, the DNAmethylation of cells in a sample obtained or provided from a subject isdetected. In embodiments, the sample comprises DCIS-containing cancercells isolated by a laser capture procedure from a biopsy. Inembodiments, the sample comprises a DCIS cancer cell proliferation thatis substantially free of normal cells. For example, at least about 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more of thecells in the DCIS cancer cell proliferation are dysplastic cells (e.g.,breast cancer cells such as DCIS cells). In embodiments, the DCIS cancercell proliferation comprises cancer cells isolated by a laser captureprocedure from a biopsy. In embodiments, the DCIS cancer cellproliferation is isolated by surgical excision followed by cancer cellpurification by using a laser capture procedure. In embodiments, theDCIS cancer cell proliferation is isolated directly from a subject orfrom a sample that was previously obtained from the subject. Inembodiments, the DCIS cancer cell proliferation is obtained or providedfrom a section of tissue that has been obtained or provided from thesubject. In embodiments, the tissue comprises tissue from a mammographicabnormality or lump in the breast of a subject that comprises or issuspected of comprising breast cancer cells such as DCIS cells. Inembodiments, the DCIS cancer cell proliferation is obtained or providedfrom a tissue section, e.g. a slice or section or a sample that has beenstained for DNA and protein detection such as a slice or section thathas been mounted on a slide. In embodiments, the DCIS cancer cellproliferation is isolated or captured from a paraffin-embedded sampleslide using, e.g., a laser. In embodiments, DNA methylation of at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, 75, 100, 125, or 140,or 1-50, 1-100, 1-140, 50-100, or 50-140 (inclusive) of the sites inTable 1 identifies the DCIS of a subject as IC-DCIS or II-DCIS.

Method of Detection Methylation Status of a Ductal Carcinoma DNA

In an aspect, provided herein is a method for detecting the presence,absence, and/or level of methylation at one or more sites set forth inTable 1. In embodiments, the level of methylation at a site set forth inTable 1 is detected. In embodiments, determining the level ofmethylation comprises bisulfite salt treatment, methyl-sensitive cutcounting, and/or any method for a single base DNA methylation detection.

In an aspect, included herein is method of detecting methylation orunmethylation of a DCIS cell DNA molecule of a subject. In embodiments,the method comprises (i) contacting an isolated DCIS cancer cellproliferation DNA molecule from the subject with a bisulfite saltthereby forming a reacted DCIS cancer cell proliferation DNA molecule;and (ii) detecting the presence or absence of uracil in the reacted DCIScancer cell proliferation DNA molecule at a methylation site set forthin Table 1, thereby detecting methylation or unmethylation of the DCIScancer cell proliferation DNA molecule of the subject.

In embodiments, the present disclosure provides a method of detectingmethylation or unmethylation of a breast lump DNA molecule of a subject,the method including: (i) isolating DNA from cancer cells obtained froma mammographic or Mill ductal abnormality, a breast nodule, a tumor, ora lump of said subject (e.g., a DCIS cancer cell proliferation ortissue) thereby forming an isolated DNA molecule, (ii) contacting saidisolated DNA molecule with a bisulfite salt (such as sodium bisulfite)thereby forming a reacted DNA molecule, (iii) detecting the presence orabsence of uracil in said reacted DNA molecule at a methylation site setforth in Table 1, thereby detecting methylation or unmethylation of saidDNA molecule of said subject.

In an aspect, provided herein is a method of detecting methylation orunmethylation of a DCIS cancer cell proliferation DNA of a subject. Themethod includes: (i) isolating a DCIS cancer cell proliferation DNAmolecule from a DCIS cancer cell proliferation of the subject therebyforming an isolated DCIS cancer cell proliferation DNA molecule, (ii)contacting the isolated DCIS cancer cell proliferation DNA molecule witha bisulfite salt (such as sodium bisulfite) thereby forming a reactedDCIS cancer cell proliferation DNA molecule, (iii) detecting thepresence or absence of uracil in the reacted DCIS cancer cellproliferation DNA molecule at a methylation site set forth in Table 1,thereby detecting methylation or unmethylation of the DCIS cancer cellproliferation DNA molecule of the subject. In embodiments, contactingthe isolated DCIS cancer cell proliferation DNA with a bisulfite saltcomprises adding a solution comprising the bisulfite salt to a solutioncomprising the isolated single stranded DNA.

In embodiments, the method of detecting methylation or unmethylation ofa DCIS DNA molecule of a subject, includes determining alteration (e.g.,compared to normal or II-DCIS cancer cell proliferation DNA) inmethylation at a plurality of methylation sites set forth in Table 1.

Also provided herein is a method of detecting methylation orunmethylation of a plurality of DCIS cancer cell proliferation DNAmolecules of a subject. In embodiments, the method comprises (i)contacting a plurality of isolated DCIS cancer cell proliferation DNAmolecules from the subject with a bisulfite salt thereby forming aplurality of reacted DCIS cancer cell proliferation DNA molecules; and(ii) detecting the level of uracil in the plurality of reacted DCIScancer cell proliferation DNA molecules at a plurality of methylationsites set forth in Table 1, thereby detecting the level of methylationor unmethylation in the plurality of DCIS cancer cell proliferation DNAmolecules of the subject.

In an aspect, provided herein is a method of detecting methylation orunmethylation of a plurality of DCIS cancer cell proliferation DNAmolecules of a subject comprising (i) isolating a plurality of DCIScancer cell proliferation DNA molecules from the DCIS cancer cellproliferation of the subject thereby forming a plurality of isolatedDCIS cancer cell proliferation DNA molecules, (ii) contacting theplurality of isolated DCIS cancer cell proliferation DNA molecules withthe bisulfite salt thereby forming a plurality of reacted DCIS cancercell proliferation DNA molecules, (iii) detecting the level of reactedDCIS cancer cell proliferation DNA molecules in the plurality of reactedDCIS cancer cell proliferation DNA molecules having a uracil at aplurality of methylation sites set forth in Table 1, thereby detectingthe level of methylation or unmethylation in the plurality of DCIScancer cell proliferation DNA molecules of the subject.

In an aspect, provided herein is a method of detecting methylation orunmethylation of a plurality of DCIS cancer cell proliferation DNAmolecules of a subject comprising (i) isolating a plurality of DCIScancer cell proliferation DNA molecules from the DCIS cancer cellproliferation of the subject thereby forming a plurality of isolatedDCIS cancer cell proliferation DNA molecules, (ii) contacting theplurality of isolated DCIS cancer cell proliferation DNA molecules withthe bisulfite salt thereby forming a plurality of reacted DCIS cancercell proliferation DNA molecules, (iii) plurality of reacted DCIS cancercell proliferation DNA molecules having a uracil at a plurality ofmethylation sites set forth in Table 1, thereby detecting methylation orunmethylation in the plurality of DCIS cancer cell proliferation DNAmolecules of the subject.

In an aspect, provided herein is a method of detecting methylation orunmethylation of a DCIS cancer cell proliferation DNA molecule of asubject. The method includes: (i) contacting an isolated DCIS cancercell proliferation DNA molecule from the subject with a bisulfite salt(such as sodium bisulfite) thereby forming a reacted DCIS cancer cellproliferation DNA molecule, (ii) amplifying the reacted DCIS cancer cellproliferation DNA molecule thereby forming a reacted DCIS cancer cellproliferation DNA amplicon molecule, (iii) detecting the presence orabsence of thymidine in a reacted DCIS cancer cell proliferation DNAamplicon molecule at a methylation site set forth in Table 1, therebydetecting methylation or unmethylation of the DCIS cancer cellproliferation DNA molecule of the subject. In embodiments, contactingthe isolated DCIS cancer cell proliferation DNA with a bisulfite saltcomprises adding a solution comprising the bisulfite salt to a solutioncomprising the isolated single stranded DNA.

In an aspect, provided herein is a method of detecting methylation orunmethylation of a DCIS cancer cell proliferation DNA molecule of asubject. The method includes: (i) isolating a DCIS cancer cellproliferation DNA molecule from a DCIS cancer cell proliferation of thesubject thereby forming an isolated DCIS cancer cell proliferation DNAmolecule, (ii) contacting the isolated DCIS cancer cell proliferationDNA molecule with a bisulfite salt (such as sodium bisulfite) therebyforming a reacted DCIS cancer cell proliferation DNA molecule, (iii)amplifying the reacted DCIS cancer cell proliferation DNA moleculethereby forming a reacted DCIS cancer cell proliferation DNA ampliconmolecule, (iv) detecting the presence or absence of thymidine in areacted DCIS cancer cell proliferation DNA amplicon molecule at amethylation site set forth in Table 1, thereby detecting methylation orunmethylation of the DCIS cancer cell proliferation DNA molecule of thesubject. In embodiments, contacting the isolated DCIS cancer cellproliferation DNA with a bisulfite salt comprises adding a solutioncomprising the bisulfite salt to a solution comprising the isolatedsingle stranded DNA.

In an aspect, provided herein is a method of detecting methylation orunmethylation of a plurality of DCIS cancer cell proliferation DNAmolecules of a subject comprising (i) contacting a plurality of isolatedcancer DCIS cancer cell proliferation DNA molecules from a subject witha bisulfite salt thereby forming a plurality of reacted DCIS cancer cellproliferation DNA molecules, (ii) amplifying the plurality of reactedDCIS cancer cell proliferation DNA molecules thereby forming a pluralityof reacted DCIS cancer cell proliferation DNA amplicon molecules, (iii)detecting one or more DCIS cancer cell proliferation DNA ampliconmolecules within the plurality of reacted DCIS cancer cell proliferationDNA amplicon molecules having a thymidine at a methylation site setforth in Table 1, thereby detecting methylation or unmethylation of theplurality of DCIS cancer cell proliferation DNA molecules of thesubject.

In an aspect, provided herein is a method of detecting methylation orunmethylation of a plurality of DCIS cancer cell proliferation DNAmolecules of a subject comprising (i) isolating a plurality of DCIScancer cell proliferation DNA molecules from the DCIS cancer cellproliferation of the subject thereby forming a plurality of isolatedDCIS cancer cell proliferation DNA molecules, (ii) contacting theplurality of isolated DCIS cancer cell proliferation DNA molecules witha bisulfate salt thereby forming a plurality of reacted DCIS cancer cellproliferation DNA molecules, (iii) amplifying the plurality of reactedDCIS cancer cell proliferation DNA molecules thereby forming a pluralityof reacted DCIS cancer cell proliferation DNA amplicon molecules, (iv)detecting one or more DCIS cancer cell proliferation DNA ampliconmolecules within the plurality of reacted DCIS cancer cell proliferationDNA amplicon molecules having a thymidine at a methylation site setforth in Table 1, thereby detecting methylation or unmethylation of theplurality of DCIS cancer cell proliferation DNA molecules of thesubject.

In embodiments, detecting one or more DCIS cancer cell proliferation DNAamplicon molecules comprises detecting the level of one or more one ormore DCIS cancer cell proliferation DNA amplicon molecules. Inembodiments, detecting one or more DCIS cancer cell proliferation DNAamplicon molecules comprises detecting the level of reacted DCIS cancercell proliferation DNA amplicon molecules in the plurality of reactedDCIS cancer cell proliferation DNA amplicon molecules having a thymidineat a methylation site set forth in Table 1, thereby detecting the levelof methylation or unmethylation in the plurality of DCIS cancer cellproliferation DNA molecules of the subject.

In embodiments, detecting a level includes determining the number (e.g.quantitating) or molecules having, e.g., a thymidine or a uracil. Inembodiments, detecting a level includes detecting the portion orproportion of a population or plurality of molecules having, e.g., athymidine or a uracil.

In embodiments, the isolated DCIS cancer cell proliferation DNA sampleis treated with a bisulfite reagent, e.g., a bisulfite salt (i.e., aprocess called DNA bisulfite conversion). Non-limiting examples ofbisulfite salts include sodium bisulfite, potassium bisulfite, ammoniumbisulfite, magnesium bisulfite, sodium metabisulfite, potassiummetabisulfite, ammonium metabisulfite and magnesium metabisulfite.Bisulfite salts such as sodium bisulfite or ammonium bisulfite canconvert cytosine to uracil and leave 5-methylcytosine (5-mC) the same.Thus after bisulfite treatment methylated cytosine in the DNA remainsthe same and unmodified cytosines will be changed to uracil. Thebisulfate treatment can be performed by using the methods disclosedherein or in the art, and/or with commercial kits such as the BisulFlashDNA Modification Kit (EpiGentek) and Imprint DNA Modification Kit(Sigma). For achieving the optimal bisulfite conversion, the bisulfitereaction should be carried out in an appropriate concentration ofbisulfite reagents, appropriate temperature and appropriate reactiontime period. A reagent such as potassium chloride that reducesthermophilic DNA degradation could also be used in bisulfite treatmentso that the DNA bisulfite process can be much shorter withoutinterrupting a completed conversion of unmethylated cytosine to uraciland without a significant thermodegradation of DNA resulted fromdepurination. In embodiments, a commercially available bisulfitetreatment kit is used. A non-limiting example of such a kit is EZ DNAMethylation-Gold™ Kit (Zymo Research, Irvine, Calif., USA).

In embodiments, once DNA bisulfite conversion is complete, DNA iscaptured, desulphonated and washed. In embodiments, thebisulfite-treated DNA can be captured by, e.g., a solid matrix selectedfrom silica salt, silica dioxide, silica polymers, magnetic beads, glassfiber, celite diatoms and nitrocellulose in the presence of highconcentrations of chaotropic or non-chaotropic salts. In embodiments,the bisulfite-treated DNA is further desulphonated with an alkalizedsolution, preferably sodium hydroxide at concentrations from 10 mM to300 mM. In embodiments, the DNA is then eluted and collected into acapped microcentrifuge tube. Non-limiting examples of elution solutionsinclude DEPC-treated water and TE buffer (10 mM Tris-HCL, pH 8.0, and 1mM EDTA).

In embodiments, the reacted DCIS cancer cell proliferation DNA resultingfrom bisulfite treatment is amplified. In embodiments, detecting thepresence or absence of uracil in reacted DCIS cancer cell proliferationDNA molecule at a methylation site comprises amplifying the reacted DCIScancer cell proliferation DNA molecule thereby forming a reacted DCIScancer cell proliferation DNA amplicon molecule, and detecting thepresence or absence of thymidine in a reacted DCIS cancer cellproliferation DNA amplicon molecule at the methylation site. Inembodiments, a polymerase chain reaction (PCR) method is used foramplifying the reacted DCIS cancer cell proliferation DNA. PCR methodsare known to those of ordinary skill in the art. In general, the PCRreactions can be set up by adding sample, dNTPs, and appropriatepolymerase such as Taq polymerase, primers, and a buffer.

In embodiments, the method of detecting methylation or unmethylation ofa DCIS cancer cell proliferation DNA of a subject, includes detectingmethylation or unmethylation at a plurality of methylation sites setforth in Table 1. In embodiments, the plurality of methylation sitescomprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80,90, 100, 110, 120, 130, or 140 methylation sites. In embodiments, theplurality of methylation sites comprises less than about 140, 130, 120,110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 methylation sites. Inembodiments, the plurality of methylation sites is about 2, 3, 4, 5, 10,25, 50, 75, 80, 85, 90, 100, 110, 120, 130, or 140 methylation sites. Inembodiments, the plurality of methylation sites includes one, two, ormore methylation sites set forth in Table 1 and no other methylationsites.

In embodiments, the method includes detecting methylation orunmethylation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, or 140 of the following sites:Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1position 91183172, Chr1 position 166853786, Chr1 position 179545096,Chr1 position 207669851, Chr1 position 237205704, Chr1 position237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3)position 14852857, Chr3 position 121903470, Chr3 position 170303393,Chr3 position 170303422, Chr3 position 170303423, Chr3 position170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5(Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177,Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6)position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7position 150038502, Chr7 position 157477232, Chr7 position 157477399,Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8position 56015232, Chr8 position 65281933, Chr8 position 145105472,Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10position 50818610, Chr10 position 77157527, Chr10 position 123778639,Chr10 position 123778640, Chr10 position 124902829, Chr10 position124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11position 9025912, Chr11 position 15963013, Chr11 position 66187593,Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12)position, Chr12 position 49726711, Chr12 position 50297756, Chr12position 50297763, Chr12 position 50297768, Chr12 position 50297774,Chr12 position 50297776, Chr12 position 50444766, Chr12 position75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13)position 28502190, Chr13 position 79181509, Chr13 position 92051154,Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14)position 29236052, Chr14 position 29236065, Chr14 position 101543886,Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15position 76630094, Chr15 position 89951787, Chromosome 16 position1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17position 35294171, Chr17 position 64831307, Chr17 position 74136562,Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18position 19745054, Chr18 position 19747206, Chr18 position 44774403,Chr18 position 55103840, Chr18 position 55106910, Chr18 position70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20position 48626669, Chr20 position 53092916, Chr20 position 59827619,Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21position 9826150, Chr21 position 9826934, and Chromosome 22 position43807517.

In embodiments, the method includes detecting methylation orunmethylation of at least 1 of the following sites: Chromosome 1 (Chr1)position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1position 166853786, Chr1 position 179545096, Chr1 position 207669851,Chr1 position 237205704, Chr1 position 237205705, Chr1 position240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3position 170303423, Chr3 position 170303424, Chr3 position 170303425,Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7(Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896,Chr7 position 71801905, Chr7 position 100946148, Chr7 position100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7position 157477232, Chr7 position 157477399, Chr7 position 157477401,Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10position 124902829, Chr10 position 124909545, Chr10 position 130085373,Chr10 position 134598235, Chr10 position 134599080, Chromosome 11(Chr11) position 1215978, Chr11 position 9025912, Chr11 position15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11position 101453451, Chromosome 12 (Chr12) position, Chr12 position49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12position 50297768, Chr12 position 50297774, Chr12 position 50297776,Chr12 position 50444766, Chr12 position 75601447, Chr12 position95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17position 3211643, Chr17 position 30244229, Chr17 position 35294171,Chr17 position 64831307, Chr17 position 74136562, Chr17 position74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18position 55103840, Chr18 position 55106910, Chr18 position 70534832,Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19)position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20position 3073503, Chr20 position 10198305, Chr20 position 23015989,Chr20 position 23016002, Chr20 position 26189258, Chr20 position48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21position 9826150, Chr21 position 9826934, and Chromosome 22 position43807517.

In embodiments, the method includes detecting methylation orunmethylation of at least 5 of the following sites: Chromosome 1 (Chr1)position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1position 166853786, Chr1 position 179545096, Chr1 position 207669851,Chr1 position 237205704, Chr1 position 237205705, Chr1 position240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3position 170303423, Chr3 position 170303424, Chr3 position 170303425,Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7(Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896,Chr7 position 71801905, Chr7 position 100946148, Chr7 position100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7position 157477232, Chr7 position 157477399, Chr7 position 157477401,Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10position 124902829, Chr10 position 124909545, Chr10 position 130085373,Chr10 position 134598235, Chr10 position 134599080, Chromosome 11(Chr11) position 1215978, Chr11 position 9025912, Chr11 position15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11position 101453451, Chromosome 12 (Chr12) position, Chr12 position49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12position 50297768, Chr12 position 50297774, Chr12 position 50297776,Chr12 position 50444766, Chr12 position 75601447, Chr12 position95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17position 3211643, Chr17 position 30244229, Chr17 position 35294171,Chr17 position 64831307, Chr17 position 74136562, Chr17 position74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18position 55103840, Chr18 position 55106910, Chr18 position 70534832,Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19)position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20position 3073503, Chr20 position 10198305, Chr20 position 23015989,Chr20 position 23016002, Chr20 position 26189258, Chr20 position48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21position 9826150, Chr21 position 9826934, and Chromosome 22 position43807517.

In embodiments, the method includes detecting methylation orunmethylation of at least 10 of the following sites: Chromosome 1 (Chr1)position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1position 166853786, Chr1 position 179545096, Chr1 position 207669851,Chr1 position 237205704, Chr1 position 237205705, Chr1 position240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3position 170303423, Chr3 position 170303424, Chr3 position 170303425,Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7(Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896,Chr7 position 71801905, Chr7 position 100946148, Chr7 position100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7position 157477232, Chr7 position 157477399, Chr7 position 157477401,Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10position 124902829, Chr10 position 124909545, Chr10 position 130085373,Chr10 position 134598235, Chr10 position 134599080, Chromosome 11(Chr11) position 1215978, Chr11 position 9025912, Chr11 position15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11position 101453451, Chromosome 12 (Chr12) position, Chr12 position49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12position 50297768, Chr12 position 50297774, Chr12 position 50297776,Chr12 position 50444766, Chr12 position 75601447, Chr12 position95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17position 3211643, Chr17 position 30244229, Chr17 position 35294171,Chr17 position 64831307, Chr17 position 74136562, Chr17 position74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18position 55103840, Chr18 position 55106910, Chr18 position 70534832,Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19)position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20position 3073503, Chr20 position 10198305, Chr20 position 23015989,Chr20 position 23016002, Chr20 position 26189258, Chr20 position48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21position 9826150, Chr21 position 9826934, and Chromosome 22 position43807517.

In embodiments, the method includes detecting methylation orunmethylation of at least 50 of the following sites: Chromosome 1 (Chr1)position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1position 166853786, Chr1 position 179545096, Chr1 position 207669851,Chr1 position 237205704, Chr1 position 237205705, Chr1 position240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3position 170303423, Chr3 position 170303424, Chr3 position 170303425,Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7(Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896,Chr7 position 71801905, Chr7 position 100946148, Chr7 position100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7position 157477232, Chr7 position 157477399, Chr7 position 157477401,Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10position 124902829, Chr10 position 124909545, Chr10 position 130085373,Chr10 position 134598235, Chr10 position 134599080, Chromosome 11(Chr11) position 1215978, Chr11 position 9025912, Chr11 position15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11position 101453451, Chromosome 12 (Chr12) position, Chr12 position49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12position 50297768, Chr12 position 50297774, Chr12 position 50297776,Chr12 position 50444766, Chr12 position 75601447, Chr12 position95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17position 3211643, Chr17 position 30244229, Chr17 position 35294171,Chr17 position 64831307, Chr17 position 74136562, Chr17 position74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18position 55103840, Chr18 position 55106910, Chr18 position 70534832,Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19)position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20position 3073503, Chr20 position 10198305, Chr20 position 23015989,Chr20 position 23016002, Chr20 position 26189258, Chr20 position48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21position 9826150, Chr21 position 9826934, and Chromosome 22 position43807517.

In embodiments, the method includes detecting methylation orunmethylation of at least 100 of the following sites: Chromosome 1(Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798,Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691,Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172,Chr1 position 166853786, Chr1 position 179545096, Chr1 position207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2)position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2position 115919663, Chr2 position 115920004, Chr2 position 118982006,Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3position 121903470, Chr3 position 170303393, Chr3 position 170303422,Chr3 position 170303423, Chr3 position 170303424, Chr3 position170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951,Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998,Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6position 85484102, Chr6 position 157557787, Chr6 position 160769248,Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7position 100946151, Chr7 position 121957003, Chr7 position 150038502,Chr7 position 157477232, Chr7 position 157477399, Chr7 position157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080,Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232,Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9)position 126780185, Chr9 position 127239956, Chr9 position 140772369,Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10position 77157527, Chr10 position 123778639, Chr10 position 123778640,Chr10 position 124902829, Chr10 position 124909545, Chr10 position130085373, Chr10 position 134598235, Chr10 position 134599080,Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11position 15963013, Chr11 position 66187593, Chr11 position 71318977,Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12position 50297768, Chr12 position 50297774, Chr12 position 50297776,Chr12 position 50444766, Chr12 position 75601447, Chr12 position95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17position 3211643, Chr17 position 30244229, Chr17 position 35294171,Chr17 position 64831307, Chr17 position 74136562, Chr17 position74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18position 55103840, Chr18 position 55106910, Chr18 position 70534832,Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19)position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20position 3073503, Chr20 position 10198305, Chr20 position 23015989,Chr20 position 23016002, Chr20 position 26189258, Chr20 position48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21position 9826150, Chr21 position 9826934, and Chromosome 22 position43807517.

In embodiments, the method includes detecting methylation orunmethylation of at least 120 of the following sites: Chromosome 1(Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798,Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691,Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172,Chr1 position 166853786, Chr1 position 179545096, Chr1 position207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2)position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2position 115919663, Chr2 position 115920004, Chr2 position 118982006,Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3position 121903470, Chr3 position 170303393, Chr3 position 170303422,Chr3 position 170303423, Chr3 position 170303424, Chr3 position170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951,Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998,Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6position 85484102, Chr6 position 157557787, Chr6 position 160769248,Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7position 100946151, Chr7 position 121957003, Chr7 position 150038502,Chr7 position 157477232, Chr7 position 157477399, Chr7 position157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080,Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232,Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9)position 126780185, Chr9 position 127239956, Chr9 position 140772369,Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10position 77157527, Chr10 position 123778639, Chr10 position 123778640,Chr10 position 124902829, Chr10 position 124909545, Chr10 position130085373, Chr10 position 134598235, Chr10 position 134599080,Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11position 15963013, Chr11 position 66187593, Chr11 position 71318977,Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12position 50297768, Chr12 position 50297774, Chr12 position 50297776,Chr12 position 50444766, Chr12 position 75601447, Chr12 position95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17position 3211643, Chr17 position 30244229, Chr17 position 35294171,Chr17 position 64831307, Chr17 position 74136562, Chr17 position74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18position 55103840, Chr18 position 55106910, Chr18 position 70534832,Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19)position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20position 3073503, Chr20 position 10198305, Chr20 position 23015989,Chr20 position 23016002, Chr20 position 26189258, Chr20 position48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21position 9826150, Chr21 position 9826934, and Chromosome 22 position43807517.

In embodiments, the method includes detecting methylation orunmethylation of each of the following sites: Chromosome 1 (Chr1)position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1position 166853786, Chr1 position 179545096, Chr1 position 207669851,Chr1 position 237205704, Chr1 position 237205705, Chr1 position240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3position 170303423, Chr3 position 170303424, Chr3 position 170303425,Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7(Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896,Chr7 position 71801905, Chr7 position 100946148, Chr7 position100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7position 157477232, Chr7 position 157477399, Chr7 position 157477401,Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10position 124902829, Chr10 position 124909545, Chr10 position 130085373,Chr10 position 134598235, Chr10 position 134599080, Chromosome 11(Chr11) position 1215978, Chr11 position 9025912, Chr11 position15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11position 101453451, Chromosome 12 (Chr12) position, Chr12 position49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12position 50297768, Chr12 position 50297774, Chr12 position 50297776,Chr12 position 50444766, Chr12 position 75601447, Chr12 position95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17position 3211643, Chr17 position 30244229, Chr17 position 35294171,Chr17 position 64831307, Chr17 position 74136562, Chr17 position74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18position 55103840, Chr18 position 55106910, Chr18 position 70534832,Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19)position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20position 3073503, Chr20 position 10198305, Chr20 position 23015989,Chr20 position 23016002, Chr20 position 26189258, Chr20 position48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21position 9826150, Chr21 position 9826934, and Chromosome 22 position43807517.

Aspects provide a method for monitoring whether the risk of IDC in asubject who has DCIS. In embodiments, a method provided herein ispracticed for a subject more than once over time to determine whetherthe DCIS is progressing from II-DCIS to IC-DCIS or from IC-DCIS to IDC.In embodiments, the methylation of one or more methylation sites in asubject becomes less indicative of II-DCIS over time. In embodiments,the methylation of one or more methylation sites in a subject becomesmore indicative of IC-DCIS over time. In embodiments, methylation orunmethylation of DCIS cancer cell proliferation DNA from a subject isassessed using a method provided herein at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more times. In embodiments, the method is repeated at leastonce every 4, 6, 8, 12 or 18 months, or at least once every 2, 3, 4, or5 more years.

In embodiments, the method includes: (i) isolating DNA from multiplecells of a DCIS cancer cell proliferation of the subject thereby forminga plurality of isolated DCIS cancer cell proliferation DNA molecules,(ii) contacting the plurality of isolated DCIS cancer cell proliferationDNA molecules with a bisulfite salt thereby forming a plurality ofreacted DCIS cancer cell proliferation DNA molecules, (iii) detectingthe proportion of DNA molecules in the plurality of reacted DCIS cancercell proliferation DNA molecules having a uracil at a methylation siteset forth in Table 1, thereby detecting methylation or unmethylation ofsaid DCIS cancer cell proliferation DNA of the subject.

The methylation of a CpG site of interest may vary between individualcells (and even between chromosome pairs of individual cells) in abiological sample. When DNA is obtained from a biological sample andtreated with a bisulfite salt to convert unmethylated cytosines touracils, the bisulfite-treated DNA will typically contain (i) aproportion of DNA molecules with a cytosine at the site of interest(indicating that the site was methylated); and (ii) a proportion of DNAmolecules with a uracil at the site of interest (indicating that thesite was unmethylated). Since a uracil at a site of interest inbisulfite-treated DNA indicates that the site was unmethylated in theuntreated DNA, a thymidine at the corresponding site in an amplicon ofthe bisulfite-treated DNA (e.g., an amplicon obtained by PCR) alsoindicates that the site was unmethylated in the untreated DNA.

In embodiments, the level of methylation at a site of interest is theproportion of bisulfite-treated DNA molecules having a cytosine ratherthan a uracil at that site of interest. In embodiments, the level ofmethylation at a site of interest is the proportion of amplicons ofbisulfite-treated DNA molecules having a cytosine rather than athymidine at that site of interest.

In embodiments, the level of unmethylation at a site of interest is theproportion of bisulfite-treated DNA molecules having a uracil ratherthan a cytosine at that site of interest. In embodiments, the level ofunmethylation at a site of interest is the proportion of amplicons ofbisulfite-treated DNA molecules having a thymidine rather than acytosine at that site of interest. In Table 1, an indicated level ofuracil is the proportion of bisulfite-treated DNA molecules having auracil rather than a cytosine at the specified methylation site. Thesame levels listed in Table 1 also apply to the thymidine levels at asite of interest in an amplicon, i.e., the proportion of amplicons(derived from the PCR amplification of bisulfite-treated DNA molecules)having a thymidine rather than a cytosine at the specified methylationsite.

The level of DNA methylation at a site of interest (e.g., a methylationsite listed in Table 1) may be determined using sequencing technology.Sequencing technology can reveal nucleotide sequence variations in aplurality of DNA molecules at a single nucleotide base resolution. Forexample, the proportions of corresponding DNA molecules having a uracil,a thymidine, and/or a cytosine at a site may be determined. Anon-limiting example of a sequencing-based method for determining themethylation level at a site of interest is described in Masser et al.(2015) Targeted DNA Methylation Analysis by Next-generation Sequencing,J Vis Exp. (96): 52488, the entire content of which is incorporatedherein by reference.

The chromosomal positions listed in Tables 1-3 relate to the humangenome that is publically accessible in the University of CaliforniaSanta Cruz (UCSC) genome browser database under accession number HG19,the entire content of which is incorporated herein by reference in itsentirety. Non-limiting information regarding the UCSC Genome Browser isprovided in Kent W J, Sugnet C W, Furey T S, Roskin K M, Pringle T H,Zahler A M, Haussler D. The human genome browser at UCSC. Genome Res.2002 June; 12(6):996-1006, the entire content of which is incorporatedherein by reference. Each methylation site of interest listed in Table 1may be located in other human genomes (e.g., within the genome of aspecific subject or group of subjects) by replacing every U and R in thecorresponding sequence with a C and then searching for the location ofthe X within a reference genome by aligning the sequence against thereference genome. For example, the methylation site of interest “X” inSEQ ID NO:1 may be located within a genome by replacing each U and R inSEQ ID NO: 1 with a C (to obtain the pre-bisulfite-modified sequencehaving an X at the site of interest) and then aligning the sequenceagainst the genome using a BLAST algorithm. Also expressly provided,disclosed, and incorporated herein is the non-bisulfite-modifiedsequence corresponding to each of SEQ ID NOS: 1-242. Thenon-bisulfite-modified sequence corresponding to each of SEQ IDNOS:1-242 is each respective sequence in which each U and R is replacedwith a C, where X is the methylation site of interest. For example, thenon-bisulfite-modified sequence corresponding to SEQ ID NO:1 providedherein is a modified version of SEQ ID NO:1 in which each U and R in SEQID NO:1 is replaced with a C, where X is the methylation site ofinterest; the non-bisulfite-modified sequence corresponding to SEQ IDNO:2 provided herein is a modified version of SEQ ID NO:2 in which eachU and R in SEQ ID NO:2 is replaced with a C, where X is the methylationsite of interest; the non-bisulfite-modified sequence corresponding toSEQ ID NO:3 provided herein is a modified version of SEQ ID NO:3 inwhich each U and R in SEQ ID NO:3 is replaced with a C, where X is themethylation site of interest, and so on.

The chromosome positions listed herein correspond to the cytosine (ofthe CpG methylation site) that is methylated on the forward strand.However, the cytosine of the reverse complement of the forward strand'sCpG methylation site may also or alternatively be methylated. At eachchromosomal position provided, methylation may be determined for theforward strand, the reverse strand, or both the forward strand and thereverse strand. In embodiments, each chromosomal position listed hereinmay refer to (i) the cytosine of the methylation site on the forwardstrand, (ii) the corresponding cytosine on the reverse strand of themethylation site, or (iii) both the cytosine of the methylation site onthe forward strand and the corresponding cytosine on the reverse strandof the methylation site.

TABLE 1 Uracil level Uracil level in invasive- in invasive- SEQ SEQcompetent DCIS competent DCIS ID NO: ID NO: Chromosomal is about aboveis about below Forward Reverse Site Chromosome position indicated level*indicated level* Strand Strand 1 chr1 4714314 N/A 74.32 1 2 2 chr111413742 76.09 N/A 3 4 3 chr1 39957798 N/A 63.81 5 6 4 chr1 46951513 N/A85.11 7 8 5 chr1 47904912 N/A 73.08 9 10 6 chr1 62660691 N/A 71.20 11 127 chr1 63785800 N/A 46.94 13 14 8 chr1 67600465 N/A 42.86 15 16 9 chr191183172 N/A 48.28 17 18 10 chr1 166853786 N/A 64.29 19 20 11 chr1179545096 N/A 88.89 21 22 12 chr1 207669851 N/A 89.29 23 24 13 chr1237205704 N/A 82.61 25 26 14 chr1 237205705 N/A 78.57 25 26 15 chr1240161215 N/A 60.00 27 28 16 chr1 240934954 78.57 N/A 29 30 17 chr108076277 N/A 75.00 31 32 18 chr10 50818610 N/A 76.30 33 34 19 chr1077157527 N/A 50.43 35 36 20 chr10 123778639 74.49 N/A 37 38 21 chr10123778640 79.31 N/A 37 38 22 chr10 124902829 N/A 67.35 39 40 23 chr10124909545 N/A 80.00 41 42 24 chr10 130085373 N/A 76.74 43 44 25 chr10134598235 N/A 34.43 45 46 26 chr10 134599080 N/A 79.31 47 48 27 chr111215978 74.47 N/A 49 50 28 chr11 9025912 N/A 65.85 51 52 29 chr1115963013 N/A 51.72 53 54 30 chr11 66187593 N/A 80.95 55 56 31 chr1171318977 N/A 51.52 57 58 32 chr11 101453451 N/A 57.89 59 60 33 chr1249726711 N/A 60.00 61 62 34 chr12 50297756 N/A 69.81 63 64 35 chr1250297763 N/A 82.98 63 64 36 chr12 50297768 N/A 81.13 63 64 37 chr1250297774 N/A 75.47 63 64 38 chr12 50297776 N/A 77.36 63 64 39 chr1250444766 N/A 54.17 65 66 40 chr12 75601447 N/A 52.00 67 68 41 chr1295941925 N/A 36.84 69 70 42 chr12 128750309 83.33 N/A 71 72 43 chr12129338355 N/A 57.14 73 74 44 chr12 129338471 N/A 60.71 75 76 45 chr1328502190 N/A 65.85 77 78 46 chr13 79181509 N/A 79.84 79 80 47 chr1392051154 N/A 70.70 81 82 48 chr13 95363553 N/A 73.91 83 84 49 chr1395363592 N/A 78.95 83 84 50 chr14 29236052 N/A 72.36 85 86 51 chr1429236065 N/A 62.60 85 86 52 chr14 101543886 N/A 92.45 87 88 53 chr1529407958 N/A 69.84 89 90 54 chr15 45403826 N/A 73.68 91 92 55 chr1576630094 N/A 62.30 93 94 56 chr15 89951787 N/A 39.68 95 96 57 chr161255253 84.62 N/A 97 98 58 chr17 3211643 82.35 N/A 99 100 59 chr1730244229 70.45 N/A 101 102 60 chr17 35294171 N/A 71.43 103 104 61 chr1764831307 N/A 56.52 105 106 62 chr17 74136562 N/A 43.48 107 108 63 chr1774865566 N/A 62.79 109 110 64 chr18 19745047 N/A 67.01 111 112 65 chr1819745054 N/A 64.10 111 112 66 chr18 19747206 N/A 80.00 113 114 67 chr1844774403 N/A 68.29 115 116 68 chr18 55103840 N/A 66.20 117 118 69 chr1855106910 N/A 50.00 119 120 70 chr18 70534832 N/A 66.67 121 122 71 chr1872880039 56.52 N/A 123 124 72 chr18 77547934 N/A 33.33 125 126 73 chr1930016170 N/A 38.89 127 128 74 chr19 30017283 N/A 60.00 129 130 75 chr1930717013 N/A 54.24 131 132 76 chr19 30719659 N/A 66.67 133 134 77 chr220870821 N/A 12.20 135 136 78 chr2 45156764 N/A 34.25 137 138 79 chr274743346 N/A 34.69 139 140 80 chr2 80549703 N/A 28.21 141 142 81 chr295989474 84.21 N/A 143 144 82 chr2 105471544 N/A 63.64 145 146 83 chr2115919663 N/A 64.44 147 148 84 chr2 115920004 N/A 79.31 149 150 85 chr2118982006 N/A 47.06 151 152 86 chr2 177001540 N/A 63.16 153 154 87 chr201294019 N/A 55.43 155 156 88 chr20 3073503 N/A 62.96 157 158 89 chr2010198305 N/A 62.44 159 160 90 chr20 23015989 N/A 80.70 161 162 91 chr2023016002 N/A 65.59 161 162 92 chr20 26189258 N/A 55.56 163 164 93 chr2048626669 N/A 36.00 165 166 94 chr20 53092916 N/A 72.13 167 168 95 chr2059827619 N/A 58.82 169 170 96 chr20 59828325 N/A 76.47 171 172 97 chr219825842 N/A 37.32 173 174 98 chr21 9826150 N/A 50.00 175 176 99 chr219826934 N/A 53.36 177 178 100 chr22 43807517 N/A 40.68 179 180 101 chr314852857 N/A 57.54 181 182 102 chr3 121903470 N/A 71.96 183 184 103 chr3170303393 N/A 68.63 185 186 104 chr3 170303422 N/A 67.65 185 186 105chr3 170303423 N/A 81.82 185 186 106 chr3 170303424 N/A 56.52 185 186107 chr3 170303425 N/A 69.77 185 186 108 chr4 44449864 N/A 78.38 187 188109 chr4 54976099 N/A 72.00 189 190 110 chr4 56023880 N/A 55.10 191 192111 chr5 71014951 N/A 65.00 193 194 112 chr5 72677229 N/A 80.82 195 196113 chr5 87981177 N/A 55.26 197 198 114 chr5 140743998 N/A 71.83 199 200115 chr5 178421786 N/A 56.25 201 202 116 chr6 41337153 N/A 57.25 203 204117 chr6 85484102 N/A 68.18 205 206 118 chr6 157557787 N/A 55.56 207 208119 chr6 160769248 N/A 75.51 209 210 120 chr7 1282082 N/A 70.45 211 212121 chr7 32467637 N/A 31.03 213 214 122 chr7 71801896 N/A 63.89 215 216123 chr7 71801905 N/A 63.89 215 216 124 chr7 100946148 N/A 55.17 217 218125 chr7 100946151 N/A 43.66 217 218 126 chr7 121957003 N/A 75.00 219220 127 chr7 150038502 N/A 84.83 221 222 128 chr7 157477232 N/A 26.45223 224 129 chr7 157477399 N/A 71.43 225 226 130 chr7 157477401 N/A92.59 225 226 131 chr8 9764011 N/A 70.91 227 228 132 chr8 11566080 N/A85.51 229 230 133 chr8 11566102 N/A 65.22 229 230 134 chr8 11566125 N/A65.22 229 230 135 chr8 56015232 N/A 53.85 231 232 136 chr8 65281933 N/A95.00 233 234 137 chr8 145105472 N/A 44.93 235 236 138 chr9 126780185N/A 85.19 237 238 139 chr9 127239956 N/A 66.96 239 240 140 chr9140772369 N/A 84.62 241 242 *Level values provided are the proportion(percentage) of reacted DCIS cancer cell proliferation DNA moleculeshaving a uracil at the methylation site of interest. When ampliconsgenerated from reacted DCIS cancer cell proliferation DNA molecules(e.g., by PCR) are used to assess the level of methylation, the valuesprovided correspond to the proportion of amplicons having a thymidine atthe nucleotide position that corresponds to the methylation site ofinterest.

In embodiments, the method of detecting methylation or unmethylation ofa DCIS cancer cell proliferation DNA of a subject detects an alterationin methylation including increase or loss of uracil level at onemethylation site or a plurality of methylation sites. In embodiments,the method of detecting methylation or unmethylation of a DCIS cancercell proliferation DNA of a subject detects an alteration in methylationincluding increase or loss of thymidine level at plurality ofmethylation sites. In embodiments, the indicated levels in Tables 1, 2,and 3 are approximate indicated levels, and include values that arewithin about 15%, about 10%, or about 5% above and below the indicatedlevels.

In embodiments, the uracil level is above a threshold as set forth inTable 2 in subjects with invasive competent DCIS cancer cellproliferations. In embodiments, the thymidine level is above a thresholdas set forth in Table 2 in subjects with invasive competent DCIS cancercell proliferations. In embodiments, the uracil level is below athreshold as set forth in Table 2 in subjects with invasive incompetentDCIS cancer cell proliferations. In embodiments, the thymidine level isbelow a threshold as set forth in Table 2 in subjects with invasiveincompetent DCIS cancer cell proliferations. In embodiments, subjectwith invasive competent DCIS are identified as at risk of IDC. Inembodiments, subject with invasive incompetent DCIS are identified asnot at risk of IDC.

TABLE 2 Methylation threshold for invasive competent DCIS Uracil levelin invasive Chromosomal competent DCIS is about Chromosome positionabove indicated level* chr1 11413742 76.09 chr1 240934954 78.57 chr10123778639 74.49 chr10 123778640 79.31 chr11 1215978 74.47 chr12128750309 83.33 *Level values provided are the proportion (percentage)of reacted DCIS cancer cell proliferation DNA molecules having a uracilat the methylation site of interest. When amplicons generated fromreacted DCIS cancer cell proliferation DNA molecules (e.g., by PCR) areused to assess the level of methylation, the values provided correspondto the proportion of amplicons having a thymidine at the nucleotideposition that corresponds to the methylation site of interest.

In embodiments, the uracil level is above a threshold as set forth inTable 3 in subjects with invasive incompetent DCIS cancer cellproliferations. In embodiments, the thymidine level is above a thresholdas set forth in Table 3 in subjects with invasive incompetent DCIScancer cell proliferations. In embodiments, the uracil level is below athreshold as set forth in Table 3 in subjects with invasive competentDCIS cancer cell proliferations. In embodiments, the thymidine level isbelow a threshold as set forth in Table 3 in subjects with invasivecompetent DCIS cancer cell proliferations.

TABLE 3 Methylation threshold for invasive-incompetent DCIS Uracil levelin invasive- Chromosomal competent DCIS is about Chromosome positionbelow indicated level* chr1 4714314 74.32 chr1 39957798 63.81 chr146951513 85.11 chr1 47904912 73.08 chr1 62660691 71.20 chr1 6378580046.94 chr1 67600465 42.86 chr1 91183172 48.28 chr1 166853786 64.29 chr1179545096 88.89 chr1 207669851 89.29 chr1 237205704 82.61 chr1 23720570578.57 chr1 240161215 60.00 chr10 8076277 75.00 chr10 50818610 76.30chr10 77157527 50.43 chr10 124902829 67.35 chr10 124909545 80.00 chr10130085373 76.74 chr10 134598235 34.43 chr10 134599080 79.31 chr119025912 65.85 chr11 15963013 51.72 chr11 66187593 80.95 chr11 7131897751.52 chr11 101453451 57.89 chr12 49726711 60.00 chr12 50297756 69.81chr12 50297763 82.98 chr12 50297768 81.13 chr12 50297774 75.47 chr1250297776 77.36 chr12 50444766 54.17 chr12 75601447 52.00 chr12 9594192536.84 chr12 129338355 57.14 chr12 129338471 60.71 chr13 28502190 65.85chr13 79181509 79.84 chr13 92051154 70.70 chr13 95363553 73.91 chr1395363592 78.95 chr14 29236052 72.36 chr14 29236065 62.60 chr14 10154388692.45 chr15 29407958 69.84 chr15 45403826 73.68 chr15 76630094 62.30chr15 89951787 39.68 chr17 35294171 71.43 chr17 64831307 56.52 chr1774136562 43.48 chr17 74865566 62.79 chr18 19745047 67.01 chr18 1974505464.10 chr18 19747206 80.00 chr18 44774403 68.29 chr18 55103840 66.20chr18 55106910 50.00 chr18 70534832 66.67 chr18 77547934 33.33 chr1930016170 38.89 chr19 30017283 60.00 chr19 30717013 54.24 chr19 3071965966.67 chr2 20870821 12.20 chr2 45156764 34.25 chr2 74743346 34.69 chr280549703 28.21 chr2 105471544 63.64 chr2 115919663 64.44 chr2 11592000479.31 chr2 118982006 47.06 chr2 177001540 63.16 chr20 1294019 55.43chr20 3073503 62.96 chr20 10198305 62.44 chr20 23015989 80.70 chr2023016002 65.59 chr20 26189258 55.56 chr20 48626669 36.00 chr20 5309291672.13 chr20 59827619 58.82 chr20 59828325 76.47 chr21 9825842 37.32chr21 9826150 50.00 chr21 9826934 53.36 chr22 43807517 40.68 chr314852857 57.54 chr3 121903470 71.96 chr3 170303393 68.63 chr3 17030342267.65 chr3 170303423 81.82 chr3 170303424 56.52 chr3 170303425 69.77chr4 44449864 78.38 chr4 54976099 72.00 chr4 56023880 55.10 chr571014951 65.00 chr5 72677229 80.82 chr5 87981177 55.26 chr5 14074399871.83 chr5 178421786 56.25 chr6 41337153 57.25 chr6 85484102 68.18 chr6157557787 55.56 chr6 160769248 75.51 chr7 1282082 70.45 chr7 3246763731.03 chr7 71801896 63.89 chr7 71801905 63.89 chr7 100946148 55.17 chr7100946151 43.66 chr7 121957003 75.00 chr7 150038502 84.83 chr7 15747723226.45 chr7 157477399 71.43 chr7 157477401 92.59 chr8 9764011 70.91 chr811566080 85.51 chr8 11566102 65.22 chr8 11566125 65.22 chr8 5601523253.85 chr8 65281933 95.00 chr8 145105472 44.93 chr9 126780185 85.19 chr9127239956 66.96 chr9 140772369 84.62 *Level values provided are theproportion (percentage) of reacted DCIS cancer cell proliferation DNAmolecules having a uracil at the methylation site of interest. Whenamplicons generated from reacted DCIS cancer cell proliferation DNAmolecules (e.g., by PCR) are used to assess the level of methylation,the values provided correspond to the proportion of amplicons having athymidine at the nucleotide position that corresponds to the methylationsite of interest.

In embodiments, the method of detecting methylation or unmethylation ofa DCIS cancer cell proliferation DNA is of a candidate breast cancerpatient. In embodiments, the subject is suspected of having IDC,invasive competent DCIS, invasive incompetent DCIS, or DCIS. Inembodiments, the subject has IDC, invasive competent DCIS, invasiveincompetent DCIS, or DCIS.

In embodiments, the method of detecting methylation or unmethylation ofa DCIS cancer cell proliferation DNA is based on the level of uracil asset forth Table 2, in which the uracil level above the thresholdidentifies the DCIS cancer cell proliferation as invasive competent. Inembodiments, the method of detecting methylation or unmethylation of aDCIS cancer cell proliferation DNA is based on a level of thymidineindicated in Table 2, in which the thymidine level above the thresholdidentifies the DCIS cancer cell proliferation as invasive competent. Inembodiments, the level is the proportion of molecules (e.g., in aplurality of reacted DCIS cancer cell proliferation DNA molecules or aplurality of reacted DCIS cancer cell proliferation DNA amplicons)having a uracil or thymidine as determined by a quantitation method.Non-limiting examples of quantitation methods include sequencing andmicroarray methods.

In embodiments, the method of detecting methylation or unmethylation ofa DCIS cancer cell proliferation DNA is based on the level of uracil asset forth Table 3, in which the uracil level above the thresholdidentifies the DCIS cancer cell proliferation as invasive incompetent.In embodiments, the method of detecting methylation or unmethylation ofa DCIS cancer cell proliferation DNA is based on a level of thymidineindicated in Table 3, in which the thymidine level above the thresholdidentifies the DCIS cancer cell proliferation as invasive incompetent.In embodiments, the level is the proportion of molecules (e.g., in aplurality of reacted DCIS cancer cell proliferation DNA molecules or aplurality of reacted DCIS cancer cell proliferation DNA amplicons)having a uracil or thymidine as determined by a quantitation method.

In embodiments, the DCIS cancer cell proliferation is a specimenobtained by laser capture procedure from a biopsy or from surgicalresection of a subject.

In embodiments, the subject has undergone lumpectomy or mastectomy,radiation therapy, chemotherapy, and administration of an active agentbefore the subject undergoes the method of detecting methylation orunmethylation of a breast cancer DNA of the present disclosure.

In embodiments, the method of the present disclosure includes adetermination of prognosis for invasive ductal carcinoma.

In embodiments, the method of detecting DNA methylation level in DNA ofbreast lump may lead to changes in therapeutic regimen for treating thesubject. In embodiments, a subject identified as having invasivecompetent DCIS with a method of the present disclosure may be treatedwith lumpectomy, mastectomy, radiation therapy, chemotherapy, hormonaltherapy (such as but not limited to tamoxifen), or targeted therapy(such as but not limited to trastuzumab and everolimus), or acombination thereof. In embodiments, a subject identified as havinginvasive incompetent DCIS with a method of the present disclosure is nottreated with lumpectomy, mastectomy, radiation therapy, chemotherapy,hormonal therapy, targeted therapy, or a combination thereof.

In embodiments, the active agent administered to a subject before orafter detecting the level of methylation or unmethylation is:trastuzumab (e.g., Herceptin®), trastuzumab emtansine (e.g., Kadcyla®),lapatinib (e.g., Tykerb®), pertuzumab (e.g., Perjeta®), bevacizumab(e.g., Avastin®), tamoxifen (e.g., Nolvadex®), exemestane Aromasin®),anastrozole Arimidex), letrozole (e.g., Femara®), doxorubicin (e.g.,Adriamycin®), epirubicin (e.g., Ellence®), cyclophosphamide (e.g.,Cytoxan®), docetaxel (e.g., Taxotere®), paclitaxel (e.g., Taxol®), nabpaclitaxel (e.g., Abraxane®), eribulin (e.g., Halaven®), everolimus(e.g., Afinitor®), palbociclib (e.g., Ibrance®), capecitabine (e.g.,Xeloda®), ixabepilone (e.g., Ixempra®), methotrexate (e.g., Trexall®),or fluorouracil (also called 5-fluorouracil or 5-FU; e.g., Adrucil®).

Method of Determining Breast Cancer or Risk of Developing InvasiveDuctal Carcinoma In Situ

Aspects further provide a method of detecting a risk of developing IDCin a subject who has DCIS. In embodiments, the method comprises

-   -   (i) contacting an isolated DCIS cancer cell proliferation DNA        molecule from the subject with a bisulfite salt thereby forming        a reacted DCIS cancer cell proliferation DNA molecule; and    -   (ii) detecting the presence or absence of uracil in the reacted        DCIS cancer cell proliferation DNA molecule at a methylation        site set forth in Table 1, thereby detecting the risk for        developing IDC in the subject.

In an aspect, provided herein is a method of diagnosing IDC or detectingrisk of IDC in a subject. The method involves:

-   -   (i) isolating a DCIS cancer cell proliferation DNA molecule from        a DCIS cancer cell proliferation of the subject thereby forming        an isolated DCIS cancer cell proliferation DNA molecule;    -   (ii) contacting the isolated DCIS cancer cell proliferation DNA        molecule with a bisulfite salt (such as sodium bisulfite)        thereby forming a reacted DCIS cancer cell proliferation DNA        molecule; and    -   (iii) detecting the presence or absence of uracil in the reacted        DCIS cancer cell proliferation DNA molecule at a methylation        site set forth in Table 1; thereby diagnosing IDC or detecting        risk of IDC in the subject.

In an aspect, provided herein is a method of diagnosing IDC or detectingrisk of IDC in a subject in need thereof, comprising (i) isolating aplurality of DCIS cancer cell proliferation DNA molecules from the DCIScancer cell proliferation of the subject thereby forming a plurality ofisolated DCIS cancer cell proliferation DNA molecules, (ii) contactingthe plurality of isolated DCIS cancer cell proliferation DNA moleculeswith the bisulfite salt thereby forming a plurality of reacted DCIScancer cell proliferation DNA molecules, (iii) detecting the level ofreacted DCIS cancer cell proliferation DNA molecules in the plurality ofreacted DCIS cancer cell proliferation DNA molecules having a uracil ata methylation site set forth in Table 1; thereby diagnosing IDC ordetecting risk of IDC in the subject.

In an aspect, provided herein is a method of diagnosing IDC or detectingrisk of IDC in a subject in need thereof, comprising (i) contacting aplurality of isolated DCIS cancer cell proliferation DNA molecules fromthe subject with a bisulfite salt thereby forming a plurality of reactedDCIS cancer cell proliferation DNA molecules; and (ii) detecting thelevel of reacted DCIS cancer cell proliferation DNA molecules in theplurality of reacted DCIS cancer cell proliferation DNA molecules havinga uracil at a plurality of methylation sites set forth in Table 1,thereby detecting the risk for IDC in the subject.

In an aspect, provided herein is a method of diagnosing IDC or detectingrisk of IDC in a subject in need thereof, comprising (i) isolating aplurality of DCIS cancer cell proliferation DNA molecules from the DCIScancer cell proliferation of the subject thereby forming a plurality ofisolated DCIS cancer cell proliferation DNA molecules, (ii) contactingthe plurality of isolated DCIS cancer cell proliferation DNA moleculeswith the bisulfite salt thereby forming a plurality of reacted DCIScancer cell proliferation DNA molecules, (iii) detecting the presence orabsence of uracil in a reacted DCIS cancer cell proliferation DNAmolecule at a methylation site set forth in Table 1, thereby detectingmethylation or unmethylation of the plurality DCIS cancer cellproliferation DNA molecules of the subject.

In an aspect, provided herein is a method of diagnosing IDC or detectingrisk of IDC in a subject in need thereof. The method includes: (i)contacting an isolated DCIS cancer cell proliferation DNA molecule fromsaid subject with a bisulfite salt (such as sodium bisulfite) therebyforming a reacted DCIS cancer cell proliferation DNA molecule, (ii)amplifying the reacted DCIS cancer cell proliferation DNA moleculethereby forming a reacted DCIS cancer cell proliferation DNA ampliconmolecule, (iii) detecting the presence or absence of thymidine in areacted DCIS cancer cell proliferation DNA amplicon molecule at amethylation site set forth in Table 1, thereby detecting methylation orunmethylation of the DCIS cancer cell proliferation DNA molecule of thesubject. In embodiments, contacting the isolated DCIS cancer cellproliferation DNA with a bisulfite salt comprises adding a solutioncomprising the bisulfite salt to a solution comprising the isolatedsingle stranded DNA.

In an aspect, provided herein is a method of diagnosing IDC or detectingrisk of IDC in a subject in need thereof. The method includes: (i)isolating a DCIS cancer cell proliferation DNA molecule from a DCIScancer cell proliferation of the subject thereby forming an isolatedDCIS cancer cell proliferation DNA molecule, (ii) contacting theisolated DCIS cancer cell proliferation DNA molecule with a bisulfitesalt (such as sodium bisulfite) thereby forming a reacted DCIS cancercell proliferation DNA molecule, (iii) amplifying the reacted DCIScancer cell proliferation DNA molecule thereby forming a reacted DCIScancer cell proliferation DNA amplicon molecule, (iv) detecting thepresence or absence of thymidine in a reacted DCIS cancer cellproliferation DNA amplicon molecule at a methylation site set forth inTable 1, thereby detecting methylation or unmethylation of the DCIScancer cell proliferation DNA molecule of the subject. In embodiments,contacting the isolated DCIS cancer cell proliferation DNA with abisulfite salt comprises adding a solution comprising the bisulfite saltto a solution comprising the isolated single stranded DNA.

In an aspect, provided herein is a method of diagnosing IDC or detectingrisk of IDC in a subject in need thereof, comprising (i) contacting aplurality of isolated DCIS cancer cell proliferation DNA molecules fromsaid subject with a bisulfite salt thereby forming a plurality ofreacted DCIS cancer cell proliferation DNA molecules, (iii) amplifyingthe plurality of reacted DCIS cancer cell proliferation DNA moleculesthereby forming a plurality of reacted DCIS cancer cell proliferationDNA amplicon molecules, (iv) detecting one or more DCIS cancer cellproliferation DNA amplicon molecules within the plurality of reactedDCIS cancer cell proliferation DNA amplicon molecules having a thymidineat a methylation site set forth in Table 1, thereby detectingmethylation or unmethylation of the plurality of DCIS cancer cellproliferation DNA molecules of the subject.

In an aspect, provided herein is a method of diagnosing IDC or detectingrisk of IDC in a subject in need thereof, comprising (i) isolating aplurality of DCIS cancer cell proliferation DNA molecules from the DCIScancer cell proliferation of the subject thereby forming a plurality ofisolated DCIS cancer cell proliferation DNA molecules, (ii) contactingthe plurality of isolated DCIS cancer cell proliferation DNA moleculeswith a bisulfite salt thereby forming a plurality of reacted DCIS cancercell proliferation DNA molecules, (iii) amplifying the plurality ofreacted DCIS cancer cell proliferation DNA molecules thereby forming aplurality of reacted DCIS cancer cell proliferation DNA ampliconmolecules, (iv) detecting one or more DCIS cancer cell proliferation DNAamplicon molecules within the plurality of reacted DCIS cancer cellproliferation DNA amplicon molecules having a thymidine at a methylationsite set forth in Table 1, thereby detecting methylation orunmethylation of the plurality of DCIS cancer cell proliferation DNAmolecules of the subject.

In embodiments, detecting one or more DCIS cancer cell proliferation DNAamplicon molecules comprises detecting the level of one or more one ormore DCIS cancer cell proliferation DNA amplicon molecules. Inembodiments, detecting one or more DCIS cancer cell proliferation DNAamplicon molecules comprises detecting the level of reacted DCIS cancercell proliferation DNA amplicon molecules in the plurality of reactedDCIS cancer cell proliferation DNA amplicon molecules having a thymidineat a methylation site set forth in Table 1, thereby detecting the levelof methylation or unmethylation in the plurality of DCIS cancer cellproliferation DNA molecules of the subject.

In embodiments, detecting a level includes determining the number (e.g.quantitating) or molecules having, e.g., a thymidine or a uracil. Inembodiments, detecting a level includes detecting the portion orproportion of a population or plurality of molecules having, e.g., athymidine or a uracil.

In embodiments, contacting the isolated DCIS cancer cell proliferationDNA with a bisulfite salt comprises adding a solution comprising thebisulfite salt to a solution comprising the isolated DCIS cancer cellproliferation DNA.

In embodiments, the method of determining diagnosing IDC or detectingrisk of IDC in a subject includes selecting a subject that has or is atrisk for having or developing diagnosing IDC or detecting risk of IDC.In embodiments, the subject (a) is a woman; (b) is about 30 to about 75years old; (c) has at least one mutant breast cancer 1 (BRCA1), breastcancer 2 (BRCA2), Partner and localizer of BRCA2 (PALB2), phosphataseand tensin homolog (PTEN), or p53 allele; (d) has a parent, sibling, orchild who has been diagnosed with breast cancer; (e) has had thenon-cancerous breast diseases atypical ductal hyperplasia or lobularcarcinoma in situ; (f) has had previous radiation treatment to the chestor a breast before the age of 30; (g) has received a combination hormonetherapy with estrogen and progestin for at least five years; and/or (h)has or has had breast cancer.

In embodiments, the method includes detecting methylation orunmethylation at a plurality of methylation sites set forth in Table 1.In embodiments, the plurality of methylation sites comprises at leastabout 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120,130, or 140 methylation sites. In embodiments, the plurality ofmethylation sites comprises less than about 140, 130, 120, 110, 100, 90,80, 70, 60, 50, 40, 30, 20, or 10 methylation sites. In embodiments, theplurality of methylation sites is about 2, 3, 4, 5, 10, 20, 30, 40, 50,60, 70, 80, 80, 90, 100, 110, 120, 130, or 140 methylation sites. Inembodiments, the plurality of methylation sites includes one, two, ormore methylation sites set forth in Table 1 and no other methylationsites.

In embodiments, a method provided herein is practiced for a subject morethan once over time. In embodiments, methylation or unmethylation ofDCIS cancer cell proliferation DNA from a subject is assessed using amethod provided herein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moretimes. In embodiments, the method is repeated at least once every 4, 6,8, 12 or 18 months, or at least once every 2, 3, 4, or 5 more years.

In embodiments, the method of diagnosing IDC or detecting risk of IDC ina subject in need thereof includes determining alteration in methylationat a plurality of methylation sites set forth in Table 1. Inembodiments, the method comprises: (i) isolating DNA from multiple cellsof a DCIS cancer cell proliferation of the subject thereby forming aplurality of isolated DCIS cancer cell proliferation DNA molecules, (ii)contacting the plurality of isolated DCIS cancer cell proliferation DNAmolecules with a bisulfate salt thereby forming a plurality of reactedDCIS cancer cell proliferation DNA molecules, (iii) detecting theproportion of DNA molecules in the plurality of reacted DCIS cancer cellproliferation DNA molecules having a uracil at a methylation site setforth in Table 1.

In embodiments, the method of diagnosing IDC or detecting risk of IDC ina subject in need thereof includes alteration, i.e., increase or loss ofuracil level at plurality of methylation sites. In embodiments, themethod of determining a diagnosing IDC or detecting risk of IDC in asubject in need thereof includes alteration, i.e., increase or loss ofthymidine level at plurality of methylation sites.

In embodiments, the method of diagnosing IDC or detecting risk of IDC ina subject in need thereof includes determining a uracil level which isabove a threshold as set forth in Table 2. In embodiments, the method ofdiagnosing IDC or detecting risk of IDC in a subject in need thereofincludes determining a thymidine level which is above a thresholdindicated in Table 2. In embodiments, the level is the proportion ofmolecules (e.g., in a plurality of reacted DCIS cancer cellproliferation DNA molecules or a plurality of reacted DCIS cancer cellproliferation DNA amplicons) having a uracil or thymidine as determinedby a quantitation method.

In embodiments, the method of diagnosing IDC or detecting risk of IDC ina subject in need thereof includes determining a uracil level which isbelow a threshold as set forth in Table 3. In embodiments, the method ofdiagnosing IDC or detecting risk of IDC in a subject in need thereofincludes determining a thymidine level which is below a thresholdindicated in Table 3. In embodiments, the level is the proportion ofmolecules (e.g., in a plurality of reacted DCIS cancer cellproliferation DNA molecules or a plurality of reacted DCIS cancer cellproliferation DNA amplicons) having a uracil or thymidine as determinedby a quantitation method.

In embodiments, the method of diagnosing IDC or detecting risk of IDCinvolves a candidate IDC patient. In embodiments, the subject issuspected of having IDC or IC-DCIS. In embodiments, the subject hasIC-DCIS.

In embodiments, the method of diagnosing IDC or detecting risk of IDC ina subject in need thereof includes determining a uracil level in which athreshold above the threshold set forth in Table 2 identifies the DCIScancer cell proliferation as IC-DCIS or IDC. In embodiments, the methodof diagnosing IDC or detecting risk of IDC in a subject in need thereofincludes determining a thymidine level in which a threshold above thethreshold indicated in Table 2 identifies the DCIS cancer cellproliferation as a IC-DCIS or IDC. In embodiments, the level is theproportion of molecules (e.g., in a plurality of reacted DCIS cancercell proliferation DNA molecules or a plurality of reacted DCIS cancercell proliferation DNA amplicons) having a uracil or thymidine asdetermined by a quantitation method.

In embodiments, the method of diagnosing IDC or detecting risk of IDC ina subject in need thereof includes determining a uracil level in which athreshold above the threshold set forth in Table 3 identifies the DCIScancer cell proliferation as II-DCIS. In embodiments, the method ofdetermining a diagnosing IDC or detecting risk of IDC in a subject inneed thereof includes determining a thymidine level in which a thresholdabove the threshold indicated in Table 3 identifies the DCIS cancer cellproliferation as II-DCIS. In embodiments, the level is the proportion ofmolecules (e.g., in a plurality of reacted DCIS cancer cellproliferation DNA molecules or a plurality of reacted DCIS cancer cellproliferation DNA amplicons) having a uracil or thymidine as determinedby a quantitation method.

In embodiments, the method of diagnosing IDC or detecting risk of IDC ina subject in need thereof includes determining a uracil level in DNA ofa DCIS cancer cell proliferation specimen obtained by biopsy or bysurgical resection of a subject. In embodiments, the method ofdiagnosing IDC or detecting risk of IDC in a subject in need thereofincludes determining a thymidine level in DNA of a DCIS cancer cellproliferation specimen obtained by biopsy or by surgical resection of asubject.

Aspects provide methods of determining a prognosis or selecting atreatment for a subject comprising detecting IDC or a risk of IDC in thesubject. In embodiments, the subject has previously undergone treatmentfor breast cancer. In embodiments, the subject was in remission afterthe previous treatment for breast cancer. In embodiments, breast cancertissue has been resected from the subject. In embodiments, breast cancertissue has been resected from the subject, but the subject is not inremission for breast cancer.

In embodiments, the method of diagnosing IDC or detecting risk of IDCmay lead to a prognostic assessment of the subject. In embodiments, themethod of diagnosing IDC or detecting risk of IDC may lead to a changeor a particular choice or set of choices in the therapeutic regimen fortreating the subject. For example, a diagnosis of IDC of risk of IDC maylead to a different treatment regimen (such as surgery, hormone therapy,radiation therapy, chemotherapy, targeted therapy, or a combinationthereof) compared to a subject who is not diagnosed with IDC or a riskof IDC (such as monitoring or a treatment other than surgery, hormonetherapy, radiation therapy, chemotherapy, or targeted therapy). Inembodiments, a method of diagnosing IDC or detecting risk of IDC maylead to a determination of susceptibility (i.e., likelihood to respond)to treatment such as surgery, hormone therapy, radiation therapy,chemotherapy, targeted therapy, or a combination thereof. In embodimentsa subject identified as having IDC or IDC risk may be treated withsurgery, hormone therapy, radiation therapy, chemotherapy, targetedtherapy, or any combination thereof. In embodiments, a subjectidentified as having IDC or being at risk of developing IDC according toa method disclosed herein is advised and/or directed to receiveadditional screening and/or treatment for breast cancer.

In embodiments, a subject having IDC or at risk of developing IDS isadministered an active agent such as trastuzumab, trastuzumab emtansine,lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole,letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel,paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib,capecitabine, ixabepilone, methotrexate, or fluorouracil.

In embodiments, the method of determining an invasive ductal carcinomamay lead to changes in therapeutic regimen (e.g., treatment and/or dose)for treating the subject. In embodiments a subject identified as havingIDC with a method disclosed herein may be treated with trastuzumab,trastuzumab emtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen,exemestane, anastrozole, letrozole, doxorubicin, epirubicin,cyclophosphamide, docetaxel, paclitaxel, nab paclitaxel, eribulin,everolimus, palbociclib, capecitabine, ixabepilone, methotrexate, orfluorouracil.

In embodiments, the active agent administered to a subject afterdetermining IDC is: trastuzumab, trastuzumab emtansine, lapatinib,pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole,doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nabpaclitaxel, eribulin, everolimus, palbociclib, capecitabine,ixabepilone, methotrexate, or fluorouracil.

Method of Treating Breast Cancer

The present disclosure provides a method of treating breast cancer in asubject by administering to the subject an active agent for treatingbreast cancer (such as IDC or IC-DCIS), in which the subject isidentified for treatment by a method disclosed herein.

The present disclosure provides a method of treating breast cancer in asubject by administering to the subject an active agent for treatingbreast cancer (such as IDC or IC-DCIS), in which the subject isidentified for treatment by a method including contacting an isolatedbreast cellular proliferation DNA with sodium bisulfite thereby forminga reacted breast cellular proliferation DNA; and detecting the presenceor absence of uracil in the reacted breast cellular proliferation DNA ata methylation site set forth in Table 1; thereby determining ductalcarcinoma invasion in the subject. In embodiments, the DCIS is notpresent as a mass or a lump. In embodiments, the DCIS is found as amammographic abnormality, such as a microcalcification in a ductalpattern.

In an aspect, provided herein is a method of treating breast cancer in asubject by administering to the subject an active agent for treatingbreast cancer (such as IDC or IC-DCIS), in which the subject isidentified for treatment by a method including isolating DNA from abreast cellular proliferation of the subject thereby forming isolatedbreast cellular proliferation DNA; contacting the isolated breastcellular proliferation DNA with sodium bisulfite thereby forming areacted breast cellular proliferation DNA; and detecting the presence orabsence of uracil in the reacted breast cellular proliferation DNA at amethylation site set forth in Table 1; thereby determining ductalcarcinoma invasion in the subject.

In embodiments, the method of treating a ductal carcinoma in a subjectin need thereof includes determining alteration in methylation at aplurality of methylation sites set forth in Table 1.

In embodiments, the method of treating a ductal carcinoma in a subjectin need thereof includes alteration which includes increase or loss ofuracil level at plurality of methylation sites.

In an aspect, included herein is a method of treating breast cancer in asubject by administering to the subject an active agent for treatingbreast cancer, in which the subject is identified for treatment by amethod including contacting an isolated DCIS cancer cell proliferationDNA molecule from the subject with a bisulfite salt (such as sodiumbisulfite) thereby forming a reacted DCIS cancer cell proliferation DNAmolecule; and detecting the presence or absence of uracil in the reactedDCIS cancer cell proliferation DNA molecule at a methylation site setforth in Table 1; thereby detecting the breast cancer in the subject. Inembodiments, contacting the isolated DCIS cancer cell proliferation DNAwith a bisulfite salt comprises adding a solution comprising thebisulfite salt to a solution comprising the isolated DCIS cancer cellproliferation DNA.

Also provided herein is a method of treating breast cancer in a subjectby administering to the subject an active agent for treating breastcancer, in which the subject is identified for treatment by a methodincluding isolating a DCIS cancer cell proliferation DNA molecule from aDCIS cancer cell proliferation of the subject thereby forming anisolated DCIS cancer cell proliferation DNA molecule; contacting theisolated DCIS cancer cell proliferation DNA molecule with a bisulfitesalt (such as sodium bisulfite) thereby forming a reacted DCIS cancercell proliferation DNA molecule; and detecting the presence or absenceof uracil in the reacted DCIS cancer cell proliferation DNA molecule ata methylation site set forth in Table 1; thereby detecting the breastcancer in the subject. In embodiments, contacting the isolated DCIScancer cell proliferation DNA with a bisulfite salt comprises adding asolution comprising the bisulfite salt to a solution comprising theisolated DCIS cancer cell proliferation DNA.

In embodiments, the method includes detecting methylation orunmethylation at a plurality of methylation sites set forth in Table 1.In embodiments, the plurality of methylation sites comprises at leastabout 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120,130, or 140 methylation sites. In embodiments, the plurality ofmethylation sites comprises less than about 140, 130, 120, 110, 100, 90,80, 70, 60, 50, 40, 30, 20, or 10 methylation sites. In embodiments, theplurality of methylation sites is about 2, 3, 4, 5, 10, 20, 30, 40, 50,60, 70, 80, 80, 90, 100, 110, 120, 130, or 140 methylation sites. Inembodiments, the plurality of methylation sites includes two or moremethylation sites set forth in Table 1 and no other methylation sites.

In embodiments, a method provided herein is practiced for a subject morethan once over time. In embodiments, methylation or unmethylation ofDCIS cancer cell proliferation DNA from a subject is assessed using amethod provided herein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moretimes. In embodiments, the method is repeated at least once every 4, 6,8, 12 or 18 months, or at least once every 2, 3, 4, or 5 more years.

In embodiments, the method of treating breast cancer in a subject inneed thereof includes determining alteration in methylation at aplurality of methylation sites set forth in Table 1.

In embodiments, the method of treating breast cancer in a subject inneed thereof includes alteration which includes increase or loss ofuracil level at plurality of methylation sites.

In embodiments, the method of treating breast cancer in a subject inneed thereof includes determining a uracil level which is above athreshold as set forth in Table 2. In embodiments, the method oftreating breast cancer in a subject in need thereof includes determininga thymidine level which is above a threshold indicated in Table 2. Inembodiments, the level is the proportion of molecules (e.g., in aplurality of reacted DCIS cancer cell proliferation DNA molecules or aplurality of reacted DCIS cancer cell proliferation DNA amplicons)having a uracil or thymidine as determined by a quantitation method.

In embodiments, the method of treating breast cancer in a subject inneed thereof includes determining a uracil level which is below athreshold as set forth in Table 3. In embodiments, the method oftreating breast cancer in a subject in need thereof includes determininga thymidine level which is below a threshold indicated in Table 3. Inembodiments, the level is the proportion of molecules (e.g., in aplurality of reacted DCIS cancer cell proliferation DNA molecules or aplurality of reacted DCIS cancer cell proliferation DNA amplicons)having a uracil or thymidine as determined by a quantitation method.

In embodiments, the method of treating breast cancer includesadministering surgery, radiation therapy, chemotherapy, targetedtherapy, or hormone therapy, before the detection of IDC.

In embodiments, the method of treating breast cancer includesadministering an active agent such as trastuzumab, trastuzumabemtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane,anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide,docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus,palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil,before the detection of IDC.

Target Sites for Methylation Level of Breast Lump

In embodiments, the present disclosure includes a deoxyribonucleic acid5 to 100 nucleotides in length including a uracil-containing sequenceidentical to at least a 5 contiguous nucleotide sequence within asequence including SEQ ID NO:1 to SEQ ID NO: 242.

Included herein are about 300 bp length sequences which are surroundingthe target sites (e.g., 149 or 150 bp from each site). The sequences areafter bisulfite conversion. Therefore “C” in the non-CpG context becomes“U”, and C in the CpG context is designated as R (either “U” either“C”). The DNA strands (sense and antisense) are no longer complementaryafter bisulfite conversion. Therefore, each DNA strand are identifiedhere with their unique sequence, and designated as “forward” and“reverse”, respectively.

In embodiments, the present disclosure includes a DNA molecule whichincludes a methylation site set forth in Table 1.

Also provided herein is a DNA molecule comprising a nucleotide sequencethat is identical to 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150,150-160, 160-170, 170-180, 180-190, or 190-200 of contiguous nucleotidesequence of the sequence including a sequence of SEQ ID NO:1 to SEQ IDNO: 242. Also included is a plurality of such DNA molecules.

In embodiments, included herein is a plurality of DNA moleculescomprising methylation sites set forth in Table 1 are methylated orunmethylated. A plurality of bisulfite-converted DNA moleculescomprising methylation sites set forth in Table 1 are also included. Inembodiments, the plurality of methylation sites comprises at least about2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120, 130,or 140 methylation sites. In embodiments, the plurality of methylationsites comprises between 1-50, 50-100, 100-250, 100-300, 100-400,100-500, 100-550, 250-550, or 350-500 methylation sites (e.g.,methylation sites included herein and others). In embodiments, theplurality of methylation sites does not comprise a methylation siteother than the sites listed in Table 1.

SEQ ID NO:1 to SEQ ID NO:242 are sequences that include the target sites(i.e., methylation sites of interest). The sequences provided are asmodified after bisulfite conversion. Therefore “C” in the non-CpGcontext becomes “U”, and C in the CpG context is designated as R or X(either “U” either “C”), where X is the target site. The DNA strands(sense and antisense) are no longer complementary after bisulfiteconversion. Therefore, each DNA strand is identified with its uniquesequence, and is designated as “forward” and “reverse” respectively, inTable 1.

The sequences listed in Table 1 are provided below with their respectivesequence identification number.

TABLE 4 Sequences Listed in Table 1 SEQ ID NO: Sequence   1GGUURGRGTGGUTUAGUTURGUUUUAGGAAAUTTTTRGRGUAGUUUURGUTUUATGRGUUUUUAUUUAGTUUTTUTTRGGGGURGUUUUUTUUUUAAAGTAGUTUTURGGGTUUAUTGGGRGUUUURGTAAURGGGTRGGAAUUTRGAAXGGUTTRGRGTGUUATURGGTTAUUUTGGUAAUAUUATURGGRGGRGUURGGRGGTUUAATUUTAGUUTGRGGUUTUTUTRGAGUUTTTRGUAAGGTGGGGAURGGGARGRGAURGGGGATGGGGAAGGGGGUTGUAGGAG GUUURGUUTG  2 UAGGRGGGGUUTUUTGUAGUUUUUTTUUUUATUUURGGTRGRGTUURGGTUUUUAUUTTGRGAAAGGUTRGAGAGAGGURGUAGGUTAGGATTGGAURGURGGGRGURGURGGATGGTGTTGUUAGGGTAAURGGATGGUARGRGAAGUXGTTRGAGGTTURGAUURGGTTARGGGGGRGUUUAGTGGAUURGGAGAGUTAUTTTGGGGAGGGGGRGGUUURGAAGAAGGAUTGGGTGGGGGRGUATGGAGRGGGGGUTGRGRGAAAAGTTTUUTGGGGRGGAGUTGAG UUARGRGGGUU  3 TTTTUTTTTTUTTTTUTTTTTTUUTTAAGGUAGAGTUTGGTTUTGTRGUUUAGAGAGRGGTAAUARGATUTUAGUTUAUTGUAAUUTUTGUUTUURGGGTTUAAGUAATTTTUATGUUTUAGGUTUUTGAGTAGUTGGAGTTAUAGGUAXGTGUTAUUATGUURGGUTAATGTTTTGTATTTTTAGTAGAGAUAGGGTTTUAUUATGTTGGUTGGGUTGGTUTTGAAUTUUUTAUUTUAAGRGATUTGUTUAUUTUAGAUTUUUAAAATGUTGGGATTAUAGGTGTGAGUUAUUAU AUTG   4UAGTGTGGTGGUTUAUAUUTGTAATUUUAGUATTTTGGGAGTUTGAGGTGAGUAGATRGUTTGAGGTAGGGAGTTUAAGAUUAGUUUAGUUAAUATGGTGAAAUUUTGTUTUTAUTAAAAATAUAAAAUATTAGURGGGUATGGTAGUAXGTGUUTGTAAUTUUAGUTAUTUAGGAGUUTGAGGUATGAAAATTGUTTGAAUURGGGAGGUAGAGGTTGUAGTGAGUTGAGATRGTGTTAURGUTUTUTGGGRGAUAGAAUUAGAUTUTGUUTTAAGGAAAAAAGAAAAGA AAAAGAAAA   5RGUUUURGGUURGUUATGGURGRGRGUUURGGAURGUTUTGGUTTUTGGGUUTGARGTTGTGRGRGUTGGGRGGGGGRGGUUURGGUUTGRGAUUUURGUURGGUTGTUUUUAGRGARGTUTGGGRGRGRGRGAGRGURGGGARGTGUAGXGRGAGATUUTGGRGGTGUTRGGGUTAUURGGGRGGUUURGGUUURGRGRGUUAUURGURGUUTUURGGUTGUURGRGTURGRGURGUTUTTUATGUTGGAUUTGTAUUARGUUATGGUTGGRGARGARGARGAGGARGG RGRGUURGRG  6 RGRGGGRGRGURGTUUTRGTRGTRGTRGUUAGUUATGGRGTGGTAUAGGTUUAGUATGAAGAGRGGRGRGGARGRGGGUAGURGGGAGGRGGRGGGTGGRGRGRGGGGURGGGGURGUURGGGTAGUURGAGUAURGUUAGGATUTRGXGUTGUARGTUURGGRGUTRGRGRGRGUUUAGARGTRGUTGGGGAUAGURGGGRGGGGGTRGUAGGURGGGGURGUUUURGUUUAGRGRGUAUAARGTUAGGUUUAGAAGUUAGAGRGGTURGGGGRGRGRGGUUATGGR GGGURGGGGGRG  7 GTUAGRGRGRGUUATGGATUAAGATGATGAATRGUTGRGGARGGRGUAGATGRGGGRGGUURGRGGURGGGUUUURGGGTAGGGGTGGGAGGTGGAGGGGGURGRGGGGGGUURGGURGURGUUATTAAUTURGGAATTAGGTUTAAGUXGUUUAUUAUUUAGUUAUTGUUURGGGGAGRGUUAGURGTTGGGGRGGGAGRGGGUUUAGGATGGGGAUTGAGAUURGRGTUUUUUAUURGAAUUTGGAUUTAGUUTUUTUTGAARGUAGAGGGUAGTGGGURGUURG GAAGGGGRGGGGA  8 TUUURGUUUUTTURGGGRGGUUUAUTGUUUTUTGRGTTUAGAGGAGGUTAGGTUUAGGTTRGGGTGGGGGARGRGGGTUTUAGTUUUUATUUTGGGUURGUTUURGUUUUAARGGUTGGRGUTUUURGGGGUAGTGGUTGGGTGGTGGGXGGUTTAGAUUTAATTURGGAGTTAATGGRGGRGGURGGGUUUUURGRGGUUUUUTUUAUUTUUUAUUUUTAUURGGGGGUURGGURGRGGGURGUURGUATUTGRGURGTURGUAGRGATTUATUATUTTGATUUATGGR GRGRGUTGAU  9 UAUURGGAUUTURGARGGUUTRGGTGTTRGUAGGRGRGGGATRGGUUUUAGUTUUTGRGUUTGUUTUAGGUTRGGGUURGGGUURGGGUUURGUAGGUUTGUURGUUTTUUTGGGRGRGGAGUTGGGUTGRGUUAAAGUUTTUTARGRGGXGTUUUTGAGTUUTUURGUAGURGGUAURGRGGRGGGTUTGUUUAURGUAUTTUTGRGUUAGGGUUTUAAGARGGARGRGGGRGGTGGTGUAGGRGGRGGGGGRGURGGGGUAGGGUAGAGGUUTTUUTTUTUTATAGAU UAUATUATGG 10 UUATGATGTGGTUTATAGAGAAGGAAGGUUTUTGUUUTGUUURGGRGUUUURGURGUUTGUAUUAURGUURGRGTURGTUTTGAGGUUUTGGRGUAGAAGTGRGGTGGGUAGAUURGURGRGGTGURGGUTGRGGGAGGAUTUAGGGAXGURGRGTAGAAGGUTTTGGRGUAGUUUAGUTURGRGUUUAGGAAGGRGGGUAGGUUTGRGGGGUURGGGUURGGGUURGAGUUTGAGGUAGGRGUAGGAGUTGGGGURGATUURGRGUUTGRGAAUAURGAGGURGTRGG AGGTURGGGTG 11 ATUUTUAUTTTGTTRGUTUUTUAGTRGTUUAGGRGGATTUUTTTTTRGUUAGGTAAGGUTGGUURGGGTGUATGGGGUURGGRGTGUUUTGGGTAAGGUTGGUUUAGGRGRGTGGGGTURGGGGRGRGURGGGTAAGGUTGGGUUAGGRGXGTGGGGTURGGGGTGUUURGGGTAAGGUTGGTUUAGGGGRGTGGGGTURGGGGTGUUURGGGTAAGGUTGGTUTAGGRGRGTGGGGTURGUAGRGUUUUAAGTAAGGUTGGTUUAGGAGRGTGAGGTUUAGGGTGUUURGG ATAAGGUT  12AGUUTTATURGGGGUAUUUTGGAUUTUARGUTUUTGGAUUAGUUTTAUTTGGGGRGUTGRGGAUUUUARGRGUUTAGAUUAGUUTTAUURGGGGUAUUURGGAUUUUARGUUUUTGGAUUAGUUTTAUURGGGGUAUUURGGAUUUUARGXGUUTGGUUUAGUUTTAUURGGRGRGUUURGGAUUUUARGRGUUTGGGUUAGUUTTAUUUAGGGUARGURGGGUUUUATGUAUURGGGUUAGUUTTAUUTGGRGAAAAAGGAATURGUUTGGARGAUTGAGGAGRGA AUAAAGTGAGGAT 13 UAAGAGATTAGUAUAATAGATUTUTAAURGAGGGGAAGRGTTGUTTTTUARGUTARGRGURGTAATTAATGGTATGAATUAATTAATTTGAUTTTTATTGTGTRGAAGGAAAAAAGRGUAAUAAATGGAAURGGUAGUTGGGAGTTGTTXGTUUTUUAUUUUUTTUUUUAGGGAGGTTUUAAGGAGAUAURGGGGAATGGARGGATUAGGUTGGGURGTGGUAGAGGGAGGGTAGGAGGUAGRGAUUAGUAGRGTGGAGGGAGTUUAGAGAGUTAGUUTUTGRGGARGGRG GAATRGAAA  14TTTRGATTURGURGTURGUAGAGGUTAGUTUTUTGGAUTUUUTUUARGUTGUTGGTRGUTGUUTUUTAUUUTUUUTUTGUUARGGUUUAGUUTGATURGTUUATTUUURGGTGTUTUUTTGGAAUUTUUUTGGGGAAGGGGGTGGAGGAXGAAUAAUTUUUAGUTGURGGTTUUATTTGTTGRGUTTTTTTUUTTRGAUAUAATAAAAGTUAAATTAATTGATTUATAUUATTAATTARGGRGRGTAGRGTGAAAAGUAARGUTTUUUUTRGGTTAGAGATUTATTGTGUTAATU TUTTG  15UATGGGAAAGUAAAATTAAGGGUAAUATTGAGGAAGUTUATTAATATTTAGTTTAGAAGATGUAAAGGGTTUTTURGRGAAUUTGGAAGAGUUUUURGGGUTRGUUTUTRGURGRGGTUUUAUAUTTGUUTGAUUTUAAUUAUAUTUTUUXGGTUUAGGUUURGUTGGAGAARGTUUUAURGAUTURGGGGAUAGAAAGGURGTTTATGTAAAARGARGTTTTTUUTATTTRGUUTUUUAUUUTUATGUAAATTTTGATTTTUAAUTUTTUUAAUUUTTURGUAUUUTGAAAU AAAUUTU  16GAGGTTTGTTTUAGGGTGRGGAAGGGTTGGAAGAGTTGAAAATUAAAATTTGUATGAGGGTGGGAGGRGAAATAGGAAAAARGTRGTTTTAUATAAARGGUUTTTUTGTUUURGGAGTRGGTGGGARGTTUTUUAGRGGGGUUTGGAUXGGGAGAGTGTGGTTGAGGTUAGGUAAGTGTGGGAURGRGGRGAGAGGRGAGUURGGGGGGUTUTTUUAGGTTRGRGGAAGAAUUUTTTGUATUTTUTAAAUTAAATATTAATGAGUTTUUTUAATGTTGUUUTTAATTTTGUTTT UUUATG  17GAAUTTGRGUTUUAGGAARGAUTGRGUARGTGGRGRGGRGGTGGRGGRGRGGAGGAUUUAGGRGAAGGRGAAGGRGAAGGRGAAGGRGUAGGRGAAGGRGAAGGRGUAGGRGGRGGGAAGUTARGUUAAAGURGURGURGURGURGUTGUXGGGGTUTGUUUAUAGUUTGGUAURGGGRGGUAGRGGTGGRGGRGGRGGARGRGGUAGGTGUAGRGAURGRGAAGUURGGGRGGURGRGRGUUUTRGGAAUTUUUURGRGGUTUURGAGGTGGUAGURGRGRGUUAU TRGGUAGUUURGT 18 ARGGGGUTGURGAGTGGRGRGRGGUTGUUAUUTRGGGAGURGRGGGGGAGTTURGAGGGRGRGRGGURGUURGGGUTTRGRGGTRGUTGUAUUTGURGRGTURGURGURGUUAURGUTGURGUURGGTGUUAGGUTGTGGGUAGAUUUXGGUAGRGGRGGRGGRGGRGGUTTTGGRGTAGUTTUURGURGUUTGRGUUTTRGUUTTRGUUTGRGUUTTRGUUTTRGUUTTRGUUTTRGUUTGGGTUUTURGRGURGUUAURGURGRGUUARGTGRGUAGTRGTTUUTGGAGR GUAAGTTU  19GUAURGUTTGUUTTRGUTAGAGGAAGAAGAGARGGUUUTGAGRGUAGUATUAUUTRGGURGGTTGGAGTUTGUURGGUTGURGUUAGGGGGTGATGRGUUUTUAUUTTTUUTUUAGGATGUURGGGATUUUAAGGAGGGTRGTGGGTUTUXGRGTUUUAAUUTGGAGUUTTGGUURGGGATGATAUUUTTGGUUAURGGUAGUAGGTUTUTGGTGGUUAUUTGARGUTTGAGUATTTUTTUTGRGGAUUUTGGGAAGTUUTAUUUUTTGGAUTUATAAAATATUTTGGTTG TUUAAAGGT 20 AUUTTTGGAUAAUUAAGATATTTTATGAGTUUAAGGGGTAGGAUTTUUUAGGGTURGUAGAAGAAATGUTUAAGRGTUAGGTGGUUAUUAGAGAUUTGUTGURGGTGGUUAAGGGTATUATUURGGGUUAAGGUTUUAGGTTGGGARGXGGAGAUUUARGAUUUTUUTTGGGATUURGGGUATUUTGGAGGAAAGGTGAGGGRGUATUAUUUUUTGGRGGUAGURGGGUAGAUTUUAAURGGURGAGGTGATGUTGRGUTUAGGGURGTUTUTTUTTUUTUTAGRGAAGGU AAGRGGTGU 21 UTGURGUUTRGUURGRGGGAUTUUUTGGAGGAGUTURGRGUUUTUUTUTUUATUUTUAGAGUTGURGGGRGGUTGGAGUAGUAGRGRGGGAGRGUTAGGGGUARGGGAGRGUAGTUUUTGTGGAGTRGUTGRGGGTURGRGTGGRGTGUUXGGGGGAUUUTAAAGAURGTRGGGTGGGGGUTGAGGGRGAGGGGRGGGAUAURGGGGURGRGGGRGGGGRGUAUURGGAAUUURGAUAGUTGTGTUTTGGTGGAGUTGTGGAUTGRGUURGURGAUTUUUARGGURGG GGRGGRGUTGAA 22 TTUAGRGURGUUURGGURGTGGGAGTRGGRGGGRGUAGTUUAUAGUTUUAUUAAGAUAUAGUTGTRGGGGTTURGGGTGRGUUURGUURGRGGUUURGGTGTUURGUUUUTRGUUUTUAGUUUUUAUURGARGGTUTTTAGGGTUUUUXGGGUARGUUARGRGGAUURGUAGRGAUTUUAUAGGGAUTGRGUTUURGTGUUUUTAGRGUTUURGRGUTGUTGUTUUAGURGUURGGUAGUTUTGAGGATGGAGAGGAGGGRGRGGAGUTUUTUUAGGGAGTUURGRGGGR GAGGRGGUAG 23 TGTGGTGUTGUTTGRGUTGURGGTGGUUTGGGGTGAGAGGRGGGRGGGRGTGGGGAGGRGUURGGGRGGARGAGGAAUURGGGGUUURGUAGAGAAUTRGRGTGUAGRGUTGAGUTGRGUTGUTUTGRGRGUURGGGTURGAAGGUAGRGXGATGGGTGGGUTGAGRGRGRGAUURGGUAGGGRGGRGGGTGTAGGATUUTTUTGRGUAUTGGAGAUUUTRGUTGUTTUTGGGTAAGRGTGGAGTTUUUAGGTGUAGGGGUTTAAGTRGTGARGAGRGUAGTGGAAGGRG UAGATGUTGA 24 TUAGUATUTGRGUUTTUUAUTGRGUTRGTUARGAUTTAAGUUUUTGUAUUTGGGAAUTUUARGUTTAUUUAGAAGUAGRGAGGGTUTUUAGTGRGUAGAAGGATUUTAUAUURGURGUUUTGURGGGTRGRGRGUTUAGUUUAUUUATXGRGUTGUUTTRGGAUURGGGRGRGUAGAGUAGRGUAGUTUAGRGUTGUARGRGAGTTUTUTGRGGGGUUURGGGTTUUTRGTURGUURGGGRGUUTUUUUARGUURGUURGUUTUTUAUUUUAGGUUAURGGUAGRGUAAG UAGUAUUAUA  25GUTTUUURGRGTUUTURGGGUURGGGURGUUUTUUTUURGUAUAGTGRGGAGUAGGGAGGUUURGRGUUTRGAUUAUURGRGUURGAGRGTURGRGUUTUUTUUTURGUTUTGUAGGRGGGGAURGUURGGRGUTRGGUAUURGGUAGXGRGGUUUUUTUUAGUUUURGGUTUURGGUAGUAGAAGUAGAAGGUAGRGUUAGGGGURGURGURGURGURGAGUTURGRGGGGUTRGGGAGURGGUUURGGRGAGGAGGRGRGGAAUUATGGURGATGGGGGRGAGGG RGAAGARGAGATU 26 GATUTRGTUTTRGUUUTRGUUUUUATRGGUUATGGTTURGRGUUTUUTRGURGGGGURGGUTUURGAGUUURGRGGAGUTRGGRGGRGGRGGRGGUUUUTGGRGUTGUUTTUTGUTTUTGUTGURGGGAGURGGGGGUTGGAGGGGGURGXGUTGURGGGTGURGAGRGURGGGRGGTUUURGUUTGUAGAGRGGAGGAGGAGGRGRGGARGUTRGGGRGRGGGTGGTRGAGGRGRGGGGUUTUUUTGUTURGUAUTGTGRGGGAGGAGGGRGGUURGGGUURGGAGGARG RGGGGAAGU  27GUUUUUUAGGAUTGGRGGUUURGGGUUURGUTUUUAUUUAUUUAUUTARGUAGGGGGRGTUUTGUTUAGGUAATTUUTUURGRGRGUUURGTRGGGGAURGGGRGGGGARGGGAGAAGGAAAAGGGUUUUTGGUTURGGGAUUAGGGUTUXGGAGGGTGURGGGRGGGGAGRGGAAUAGGGAARGGGUTGGTGGRGGUUUUAAGRGGGAGGGARGGAURGAUARGRGGUUUUUTGGRGGUUTTGRGAUTRGURGAUUTGRGGAAUUTRGTRGURGUUUTUAUAGUUU RGRGGUUAURGUU 28 GGRGGTGGURGRGGGGUTGTGAGGGRGGRGARGAGGTTURGUAGGTRGGRGAGTRGUAAGGURGUUAGGGGGURGRGTGTRGGTURGTUUUTUURGUTTGGGGURGUUAUUAGUURGTTUUUTGTTURGUTUUURGUURGGUAUUUTUXGGAGUUUTGGTUURGGAGUUAGGGGUUUTTTTUUTTUTUURGTUUURGUURGGTUUURGARGGGGRGRGRGGGAGGAATTGUUTGAGUAGGARGUUUUUTGRGTAGGTGGGTGGGTGGGAGRGGGGUURGGGGURGUUAGTU UTGGGGGGU  29AAAATGTTTGGUAUUTUATTGATUATGTTTAGUTGATUATUUUTGGGGUTGGGGAGGGUAGUUAGGUUAGUUAGRGRGUAGGGUAGGGGRGGGRGGAGGAGAAGUUTGGAGGAGGUTGARGGGAAGUUTRGAGGAGGGGUAUUURGGTUXGAGGUTGGGUTGGGAARGUTGUTUUAUAGRGUTGTGAGTGGURGGGAGGAUTTAURGUUAUUAURGUTGUUTUTGGGGUATUTUUUAUURGUURGGGATGUTGURGUUTUUTTTUUAUAATGUUTGTTGATUAATTTTTTA ATUAAGAAG 30 UTTUTTGATTAAAAAATTGATUAAUAGGUATTGTGGAAAGGAGGRGGUAGUATUURGGGRGGGTGGGAGATGUUUUAGAGGUAGRGGTGGTGGRGGTAAGTUUTUURGGUUAUTUAUAGRGUTGTGGAGUAGRGTTUUUAGUUUAGUUTXGGAURGGGGTGUUUUTUUTRGAGGUTTUURGTUAGUUTUUTUUAGGUTTUTUUTURGUURGUUUUTGUUUTGRGRGUTGGUTGGUUTGGUTGUUUTUUUUAGUUUUAGGGATGATUAGUTAAAUATGATUAATGAGGTGUU AAAUATTTT  31GAGUUUAGTTRGUATUTUTAUAGRGUUAUAUATUTAGGUUAGUUUAGUTURGGTUUUAGTRGGTUTUTGRGGAGUUUUAGUUAGUTURGRGTUTTGGTUUTGGUTTGRGGGTGGUAUAUAGAUAAGRGAURGGGGTGGUURGGAGAGGTXGTGUUUUTGGUTGRGAGARGARGRGAUAGUAGGGRGUTURGGGGGURGUAGRGGRGTRGAAGGRGGTUUUTGGGGGRGGGGUTUTGGUARGAGGGGGAUUUTUURGGGTUAAGTTUAUAGAGGRGGAGGGGUUTGGGUTGR GUUUUAGAG  32UTUTGGGGRGUAGUUUAGGUUUUTURGUUTUTGTGAAUTTGAUURGGGAGGGTUUUUUTRGTGUUAGAGUUURGUUUUUAGGGAURGUUTTRGARGURGUTGRGGUUUURGGAGRGUUUTGUTGTRGRGTRGTUTRGUAGUUAGGGGUAXGAUUTUTURGGGUUAUUURGGTRGUTTGTUTGTGTGUUAUURGUAAGUUAGGAUUAAGARGRGGAGUTGGUTGGGGUTURGUAGAGAURGAUTGGGAURGGAGUTGGGUTGGUUTAGATGTGTGGRGUTGTAGAGATGRG AAUTGGGUTU 33 GRGGRGGGGGUTGUTUTGGGRGRGUUURGGGRGAAGTGRGUUUAGTUTURGGUUURGGUUUUTRGGRGRGUURGAUTTUURGGURGUUUUTGAGUUUAGUAGURGRGGGTUURGGGATRGGUTAAGAGTAGUTGUAARGUUTRGURGGAXGGAGTUUTTTUUTTTUURGGGARGUTGGGUUATGAGUTURGRGGUUAUUTGAGGUAUAGGGGAGTUTGUTRGGUUAGGAUAGUUTUUURGAAGTUURGTGUUUTRGUUTUTGUAUTGRGGGARGUUAGRGUTRGGUUUTGG RGGAGGRGT  34ARGUUTURGUUAGGGURGAGRGUTGGRGTUURGUAGTGUAGAGGRGAGGGUARGGGAUTTRGGGGAGGUTGTUUTGGURGAGUAGAUTUUUUTGTGUUTUAGGTGGURGRGGAGUTUATGGUUUAGRGTUURGGGAAAGGAAAGGAUTUXGTURGGRGAGGRGTTGUAGUTAUTUTTAGURGATUURGGGAUURGRGGUTGUTGGGUTUAGGGGRGGURGGGAAGTRGGGRGRGURGAGGGGURGGGGURGGAGAUTGGGRGUAUTTRGUURGGGGRGRGUUUAGAGUA GUUUURGURGU 35 AAATGGTGAAATATUUTUTAAAAATATGTTUUUUAAGGUUAAUTTRGRGGUTGGTAGUUUUTTURGARGUUTTTGUUTUUUAGAAAATUAUAAUAAAGRGATRGGAAATTUAGUUARGGTUURGGGAAGAAGGAGTAGUAGTGAGGUUUXGGAAUUUAUTGRGGURGAAAUTGUUATGUTUTUTTTAAUUAAAATAAAAAAGATAAGAAGAAGAAGTAAAAUUUTTTAATAUATUAAATATARGGAATTTTAATUTTTAAAGRGATAUATTGTUTATTATTTTAGTAUATGA RGTAAAUU 36 GGTTTARGTUATGTAUTAAAATAATAGAUAATGTATRGUTTTAAAGATTAAAATTURGTATATTTGATGTATTAAAGGGTTTTAUTTUTTUTTUTTATUTTTTTTATTTTGGTTAAAGAGAGUATGGUAGTTTRGGURGUAGTGGGTTUXGGGGUUTUAUTGUTAUTUUTTUTTUURGGGAURGTGGUTGAATTTURGATRGUTTTGTTGTGATTTTUTGGGAGGUAAAGGRGTRGGAAGGGGUTAUUAGURGRGAAGTTGGUUTTGGGGAAUATATTTTTAGAGGATATTTUAUUAT TT  37GTGGUAGGGGTAAGGRGUTTUUATTTGUUTGAAAGGUTUTGUUUTAUAUAAAAATAGAAAUTGGTTGUATGUATGUAAAGGATGGUTGAGTGGTAATAAUTGGAGGAAUTGRGGAUAGURGGAGGUTGRGGGGURGGAGGGTUTTTGGGTXGGTAUTUTGUUUUAURGGAGGTUUAUAGGGUUTUUTUUATUUAGGGTGGGURGGUAAGTUTGUUTATUAGRGATUTGGGUAAGUUTGUTAUTTTTTATGAUUTUAUTTGURGAAAUTUAAGUUARGUUTGGGTAAGTAUA TTUTGUAUA 38 TGTGUAGAATGTAUTTAUUUAGGRGTGGUTTGAGTTTRGGUAAGTGAGGTUATAAAAAGTAGUAGGUTTGUUUAGATRGUTGATAGGUAGAUTTGURGGUUUAUUUTGGATGGAGGAGGUUUTGTGGAUUTURGGTGGGGUAGAGTAUXGAUUUAAAGAUUUTURGGUUURGUAGUUTURGGUTGTURGUAGTTUUTUUAGTTATTAUUAUTUAGUUATUUTTTGUATGUATGUAAUUAGTTTUTATTTTTGTGTAGGGUAGAGUUTTTUAGGUAAATGGAAGRGUUTTAUU UUTGUUAU  39AUTUTGGAAGTGTGUARGGURGTTGTGUAGGGTGGATGGTGTTGAUUTTTTGAUUTGAAAAUAGTTGGGGGUTGGGGAGRGGAGGAAGGATGGRGGAAGAGAGGAAAGAGUUARGAGAAUAAUTAGGRGGGATGTAUTTTTGAGUUUTGUXGGGTGTUTURGATRGGAGTUTGGGGTTGAGATTTGGGUTGUAUTTGTUUURGGTGTGTUTUTURGGRGGAGTAUUUTGAAGGTGUARGAGGTGGGGAGUATAGGUTGAGGTGGGTAATRGGGTUUTGGATAGAAAUAUAAU UUTRGTUU  40GGARGAGGGTTGTGTTTUTATUUAGGAUURGATTAUUUAUUTUAGUUTATGUTUUUUAUUTRGTGUAUUTTUAGGGTAUTURGURGGAGAGAUAUAURGGGGAUAAGTGUAGUUUAAATUTUAAUUUUAGAUTURGATRGGAGAUAUUXGGUAGGGUTUAAAAGTAUATUURGUUTAGTTGTTUTRGTGGUTUTTTUUTUTUTTURGUUATUUTTUUTURGUTUUUUAGUUUUUAAUTGTTTTUAGGTUAAAAGGTUAAUAUUATUUAUUUTGUAUAARGGURGTGUAUAUTT UUAGAGT  41AAAGAUTTGGTTUUAGAAURGURGUAAUAAGTGGAAGRGGUAGUTUTRGGUTGAGUTGGAGGRGGUUAAUATGGRGUARGRGTRGGRGUAGAUTUTGGTGAGUATGURGUTGGTGTTURGGGAUAGTTRGUTGUTGRGRGTGURGGTGUXGRGUTRGUTRGUUTTTUURGRGURGUTUTAUTAUURGGGAAGUAAUUTUTRGGUUTTAUUTUTUTAUAAUUTATAUAAUAAGUTRGAUTAUTGAURGGUURGURGUUURGRGURGUUUUUAGUTGUURGUAGAGURGGGRG RGTAUTGTA  42TAUAGTARGRGUURGGUTUTGRGGGUAGUTGGGGGRGGRGRGGGGRGGRGGGURGGTUAGTAGTRGAGUTTGTTGTATAGGTTGTAGAGAGGTAAGGURGAGAGGTTGUTTUURGGGTAGTAGAGRGGRGRGGGAAAGGRGAGRGAGRGXGGUAURGGUARGRGUAGUAGRGAAUTGTUURGGAAUAUUAGRGGUATGUTUAUUAGAGTUTGRGURGARGRGTGRGUUATGTTGGURGUUTUUAGUTUAGURGAGAGUTGURGUTTUUAUTTGTTGRGGRGGTTUTGGAAU UAAGTUTTT 43 UUAGRGUAUAGUURGGUUAGGGGRGUUUTUUUTGURGURGURGGUUUTTTGATRGUURGRGGUURGRGGUURGUAGAUUAUAGUUAGRGUTGTGUTGGGUAGGTGGRGGUAGRGGGGRGRGGRGRGUTUAGGRGUARGGGTUUURGGGUXGRGGRGRGUTUUURGUUARGURGUAUATUAAGGUURGGURGGURGGRGGGRGUUTTUATTAGUAGUUTGAAATTATAATATTATGTTAAAGAAUAAAGUTGUTUUURGGAAAAATATGTGUTGUATATUTGAUAAAGAT AAATTGGATTA 44 TAATUUAATTTATUTTTGTUAGATATGUAGUAUATATTTTTURGGGGAGUAGUTTTGTTUTTTAAUATAATATTATAATTTUAGGUTGUTAATGAAGGRGUURGURGGURGGURGGGUUTTGATGTGRGGRGTGGRGGGGAGRGRGURGXGGUURGGGGAUURGTGRGUUTGAGRGRGURGRGUUURGUTGURGUUAUUTGUUUAGUAUAGRGUTGGUTGTGGTUTGRGGGURGRGGGURGRGGGRGATUAAAGGGURGGRGGRGGUAGGGAGGGRGUUUUTGGURGGGUTG TGRGUTGG  45URGTUURGGGUTUUTGGRGGUTGTRGUTGRGGTTUUTTUURGRGGGURGGGUUUUTTUUUTGRGUUTTRGURGUUTUUTRGRGUUTGUURGGGGUURGUAGUUTURGUAURGGGAAUURGGAGGAUURGAGGRGGGRGUAGGGGRGAAGUXGGGGURGGGGAGGGGURGUUTRGUTURGGGTTRGAGARGGAAGAAAUARGRGGRGUAGGUTURGGAGRGARGGUTURGARGGGGAUURGTTAAATAATTTATTGATGATAUAAAGRGAUTRGRGUUUAUURGGGGURGU UUURGGATTU 46 GAATURGGGGGRGGUUURGGGTGGGRGRGAGTRGUTTTGTATUATUAATAAATTATTTAARGGGTUUURGTRGGAGURGTRGUTURGGAGUUTGRGURGRGTGTTTUTTURGTUTRGAAUURGGAGRGAGGRGGUUUUTUUURGGUUUXGGUTTRGUUUUTGRGUURGUUTRGGGTUUTURGGGTTUURGGTGRGGAGGUTGRGGGUUURGGGUAGGRGRGAGGAGGRGGRGAAGGRGUAGGGAAGGGGUURGGUURGRGGGAAGGAAURGUAGRGAUAGURGUUAGGAGU URGGGARGG  47UTTGTUUAGGARGURGURGGURGGGGUTGUAAGGGAGGGGAAGGGAGGGAGGTUAGRGGURGGRGGGGTUUUUUTURGRGUUUAUURGUUURGUAUUUUURGRGRGGGUUAUTUAUURGGGUUAGUUAGARGRGGGTUUUTUUAGGGRGXGUUUTGUAUUARGURGGGUUAGAAGATGGGRGGGRGUUURGGUAGUTRGGUUAGGGGUTTGGGGTAGURGRGRGUUAUAGRGGURGRGGGUURGAAGTAAARGURGGRGGARGARGRGAGUURGTTGAGURGGGGUA GUUUUUUUAGGAG 48 UTUUTGGGGGGGUTGUUURGGUTUAARGGGUTRGRGTRGTURGURGGRGTTTAUTTRGGGUURGRGGURGUTGTGGRGRGRGGUTAUUUUAAGUUUUTGGURGAGUTGURGGGGRGUURGUUUATUTTUTGGUURGGRGTGGTGUAGGGXGRGUUUTGGAGGGAUURGRGTUTGGUTGGUURGGGTGAGTGGUURGRGRGGGGGGTGRGGGGRGGGTGGGRGRGGAGGGGGAUUURGURGGURGUTGAUUTUUUTUUUTTUUUUTUUUTTGUAGUUURGGURGGRGGRGTU UTGGAUAAG  49UTURGGUUTUATUTTUTURGTGGAGGTGUUUTTUAGUAAGTTTGUUAAUAAUAURGAGGGUUAGTGRGGTGAGGUUAUAGGGUTUURGGGUATRGTUTGGUATTRGRGGGGGRGGGGGTGURGGGUAGGGGRGAGGUUAUUARGTGURGXGTGTGURGGTGTUTUTGUTTTUTGGUTGUTUTGUTGAGTGUAGGUUAUAGGUATGAGGUTTARGUUTGURGGTAUUTGUAGUTUUUUAGTAUUATAGGGAUTGTUUUAGGGTTGTTUTRGGGGGAUAGTGAGGUAUAGGUA GGGUURGUT  50AGRGGGUUUTGUUTGTGUUTUAUTGTUUUURGAGAAUAAUUUTGGGAUAGTUUUTATGGTAUTGGGGAGUTGUAGGTAURGGUAGGRGTAAGUUTUATGUUTGTGGUUTGUAUTUAGUAGAGUAGUUAGAAAGUAGAGAUAURGGUAUAXGRGGUARGTGGTGGUUTRGUUUUTGUURGGUAUUUURGUUUURGRGAATGUUAGARGATGUURGGGAGUUUTGTGGUUTUAURGUAUTGGUUUTRGGTGTTGTTGGUAAAUTTGUTGAAGGGUAUUTUUARGGAGAAGA TGAGGURGGAG 51 UUAURGRGGUUTTTTUAUUUAAURGUUUUUTUUTGRGTGGGGGUUURGUATUUUUTGGAUTGGRGTGGGUTUTGGGGUURGATGUUUTGGUAGUUURGGUUATUTUTGURGUTGGURGRGGGTAUUTGUTUAUUTGTUUARGURGTGGUXGAUURGGGGATUAURGAUAGURGGGUUATGUUTRGAGTUURGURGUURGRGURGUTGUTRGGUTTUTUAGAAARGUAUUTUUARGURGUUAGURGGGGUTUUAGGGTUUAURGUURGGGUURGGAGGAUTGGRGAUTUU UTURGAGGTTG 52 UAAUUTRGGAGGGAGTRGUUAGTUUTURGGGUURGGGRGGTGGAUUUTGGAGUUURGGUTGGRGGRGTGGAGGTGRGTTTUTGAGAAGURGAGUAGRGGRGRGGGRGGRGGGAUTRGAGGUATGGUURGGUTGTRGGTGATUUURGGGTXGGUUARGGRGTGGAUAGGTGAGUAGGTAUURGRGGUUAGRGGUAGAGATGGURGGGGUTGUUAGGGUATRGGGUUUUAGAGUUUARGUUAGTUUAGGGGATGRGGGGUUUUUARGUAGGAGGGGGRGGTTGGGTGAAA AGGURGRGGTGG 53 ATGUTAGTGUAGTTUUUTGRGUAGURGUUAGGTGGRGTUAAGGURGURGUUTGGGGGGURGGGUAGGURGAGGUUUUTGUURGTRGUAGTUUUAGUUTRGUTUAUUAUTTGGUURGGGAGTTGGUTGRGGGUAGRGGUTTGGUAGUUTGXGGURGGGGGRGGARGGTGGGGRGGTGTGGGTTTUAGUUTUUURGGAGGGUUUTUARGGUTGAGUAAARGTTRGGGUTGATGTRGGUAAUATGRGGAATUAATTTTRGGGGAAUTUAGUAGUUAAAUUATUUAUUTTTGGGR GGGAAGUAG  54UTGUTTUURGUUUAAAGGTGGATGGTTTGGUTGUTGAGTTUUURGAAAATTGATTURGUATGTTGURGAUATUAGUURGAARGTTTGUTUAGURGTGAGGGUUUTURGGGGAGGUTGAAAUUUAUAURGUUUUAURGTURGUUUURGGUXGUAGGUTGUUAAGURGUTGUURGUAGUUAAUTUURGGGUUAAGTGGTGAGRGAGGUTGGGAUTGRGARGGGUAGGGGUUTRGGUUTGUURGGUUUUUUAGGRGGRGGUUTTGARGUUAUUTGGRGGUTGRGUAGGGAAUT GUAUTAGUAT 55 GGAGRGGTGRGGAGGUAGUUAGGUUURGUURGURGUAGURGRGGUAGURGURGGAGGATTUUTGTUUTAATATGGAGUTGGGATTUUUURGGUUURGUUUURGUUUURGGUURGRGGGGAGAUAGAGGUTGGUAGUAGGGRGGGGGGAAGXGUTRGUTTGGGGGURGGUAARGGGGGGAAGGGATGUUTAAGTGUAGAUUUAGGTUUTRGURGTGUUUUUARGTUUUTGUUTUAGTTTUUUUTTUAGTAAGGTTAATTAGUTGAGAGGGAAAUUARGAATUAUTGUAG AUTAUAGRGUTG 56 UAGRGUTGTAGTUTGUAGTGATTRGTGGTTTUUUTUTUAGUTAATTAAUUTTAUTGAAGGGGAAAUTGAGGUAGGGARGTGGGGGUARGGRGAGGAUUTGGGTUTGUAUTTAGGUATUUUTTUUUUURGTTGURGGUUUUUAAGRGAGXGUTTUUUUURGUUUTGUTGUUAGUUTUTGTUTUUURGRGGGURGGGGGRGGGGGRGGGGURGGGGGAATUUUAGUTUUATATTAGGAUAGGAATUUTURGGRGGUTGURGRGGUTGRGGRGGGRGGGGUUTGGUTGUUTURG UAURGUTUU  57RGGAGTGGAGUTTTGGGAAUUUUTRGGUUAAGUAUAGRGGTTRGAAAATAUAGUTGAAAUUUAGRGGGUUUTTAGUARGRGUUUUAGRGURGGAGUAGGGTUAGGGTUTTUTTGRGAUURGGUTURGUTUUAGATUUUUUUAGUTUTRGGXGGRGGAUURGGGURGRGTGTGAGRGRGUTTTGUAUUTUUTATUUUUAGGGTURGURGAGAGUUARGATTTTTTAUAGAAAATGAGUAATAAAGAGATTTTGTAUTGTUUTGAUTGGGGAGTUUUAGGURGRGGGGGARGG AGRGUUUUT  58AGGGGRGUTURGTUUUURGRGGUUTGGGAUTUUUUAGTUAGGAUAGTAUAAAATUTUTTTATTGUTUATTTTUTGTAAAAAATRGTGGUTUTRGGRGGAUUUTGGGGATAGGAGGTGUAAAGRGRGUTUAUARGRGGUURGGGTURGUXGURGAGAGUTGGGGGGATUTGGAGRGGAGURGGGTRGUAAGAAGAUUUTGAUUUTGUTURGGRGUTGGGGRGRGTGUTAAGGGUURGUTGGGTTTUAGUTGTATTTTRGAAURGUTGTGUTTGGURGAGGGGTTUUUAAAGUT UUAUTURG  59AAAAATAUUUTAGATTUATAAAUAAAAGUTTGUAGTTAUUUAUAGUUUAATAUAAARGAUURGARGUUUTUAUTTUTATAGUURGTGAGTUTUUUAGUUUUTAAUAGGUTRGTUTUUUUAGARGUUURGGGTGAAAAGGTTRGRGURGUXGGTGGAGAGTUUTATTGGTTUTATTTUTGTUTTUAUTUUAAGGUUUUAGGAURGGGGAGAGGTUUTGGTUTAAUTTTGGUTGTUUUARGGTUUAAGGAUUTUAGRGUTUAGUURGAAARGUTAAGUUAAGRGTGGAGAURG URGGGRGUA  60TGRGUURGGRGGTUTUUARGUTTGGUTTAGRGTTTRGGGUTGAGRGUTGAGGTUUTTGGAURGTGGGAUAGUUAAAGTTAGAUUAGGAUUTUTUUURGGTUUTGGGGUUTTGGAGTGAAGAUAGAAATAGAAUUAATAGGAUTUTUUAUXGGRGGRGRGAAUUTTTTUAUURGGGGRGTUTGGGGAGARGAGUUTGTTAGGGGUTGGGAGAUTUARGGGUTATAGAAGTGAGGGRGTRGGGTRGTTTGTATTGGGUTGTGGGTAAUTGUAAGUTTTTGTTTATGAATUTAGGG TATTTTT  61AGUUARGAUUTUTGUUUUTATGGGGTUUAGRGRGUAUTTGGGTGRGGGTGATUUUTURGGAAGTRGUTUTGUTUUTUTGGURGGGTUTUUTUUTUTTUURGGUUAUTTATAUUUTTGAGUAUUAAGAGTTRGUUUATTTAGGURGUUTUXGGGAARGGAAGGGTUUAUUUUAURGUUAGGUTUTUAAAGGGTGGGGTGGRGUUTRGGGTGGGGRGGGUAGGAGGTGGGTGAGGARGGGAGAGAAGGGGRGAGRGGGGGUARGGGGRGGGGURGGUTUAGTRGGGGTAGAT GATGAAGURG  62RGGUTTUATUATUTAUUURGAUTGAGURGGUUURGUUURGTGUUUURGUTRGUUUUTTUTUTUURGTUUTUAUUUAUUTUUTGUURGUUUUAUURGAGGRGUUAUUUUAUUUTTTGAGAGUUTGGRGGTGGGGTGGAUUUTTURGTTUUXGGAGGRGGUUTAAATGGGRGAAUTUTTGGTGUTUAAGGGTATAAGTGGURGGGAAGAGGAGGAGAUURGGUUAGAGGAGUAGAGRGAUTTURGGAGGGATUAUURGUAUUUAAGTGRGRGUTGGAUUUUATAGGGGUAG AGGTRGTGGUT 63 URGUTTGGGTAAURGUAGUUTGUUTGRGTUTUTTUUTTUUTURGRGTGGGTTUTAGUAAUATUUAUTGUAGURGGGUUAGGRGAGURGGRGRGTAUUATRGGRGRGGGGGGAGGAGAGGGURGGGUUTGGGAAGATGUTGXGGAGGAXGUTGXGGATTXGXGAGUURGGGGTAAGGRGGRGGRGUAURGUUUUUTUURGURGUTTUUUUUUUAUUURGUUUUUUAURGURGUUUTTAGUUUTUUUURGGGATGAGAGAGAGTRGRGUTGRGGAGUAAUUUUAGTGGAT GGGTURGRGGGG 64 UUURGRGGAUUUATUUAUTGGGGTTGUTURGUAGRGRGAUTUTUTUTUATUURGGGGGAGGGUTAAGGGRGGRGGTGGGGGGRGGGGTGGGGGGGAAGRGGRGGGAGGGGGRGGTGRGURGURGUUTTAUUURGGGUTXGXGAATUXGUAGXGTUUTUXGUAGUATUTTUUUAGGUURGGUUUTUTUUTUUUUURGRGURGATGGTARGRGURGGUTRGUUTGGUURGGUTGUAGTGGATGTTGUTAGAAUUUARGRGGAGGAAGGAAGAGARGUAGGUAGGUTGRGGT TAUUUAAGRGG 65 ATUTUUUUTTUTGUUTGTGATATGGRGGGUTGUATTAUAAGUTGGGRGTRGAGATAAAGRGGGGAGURGRGGGARGGRGGUUUAGRGURGGGGUUURGGGTGGGGURGGUUAGGGAGATAAGGGUUTUUUAGUUUUATGAATTATTUAUXGGUAUTGUUTUUTUTGGUTGGGRGGURGUUUTGGGRGUTGAGATTGRGTUTTUUAGAGGGGUUTGGGTAGGGTGGGAGGGGGGTURGTGGGGAAGGGUAGUAGGUTGTAGAGGGGTUTTUUTATGGGGGATGGGGAGAGG GGAGGAUTGT 66 AUAGTUUTUUUUTUTUUUUATUUUUUATAGGAAGAUUUUTUTAUAGUUTGUTGUUUTTUUUUARGGAUUUUUUTUUUAUUUTAUUUAGGUUUUTUTGGAAGARGUAATUTUAGRGUUUAGGGRGGURGUUUAGUUAGAGGAGGUAGTGUXGGTGAATAATTUATGGGGUTGGGAGGUUUTTATUTUUUTGGURGGUUUUAUURGGGGUUURGGRGUTGGGURGURGTUURGRGGUTUUURGUTTTATUTRGARGUUUAGUTTGTAATGUAGUURGUUATATUAUAGGU AGAAGGGGAGAT 67 AGGTUATUUAGUAGUAGGGUTUUARGTRGGTUTRGTRGATGUUUUAGAAGGUUAGUTUUTUUTRGAAGAGRGGUURGUAUARGTUTGRGGGGUAGTGUAGUTTGURGGTGRGGTAGTAATTGAGUAUATAGGRGAAGARGUURGGGTGUXGGTRGAAGAAGAAUTRGRGGURGUUAURGGGATGGTRGUTGGUUUTGURGURGRGGGAAUTGUAGTTGUURGRGURGUUUTRGAAGUAGURGUUTGGUURGGGGGAUAGRGGGGGRGUTUTRGGRGGRGGRGAUAGTGG AGGRGGRGARG 68 RGTRGURGUUTUUAUTGTRGURGURGURGAGAGRGUUUURGUTGTUUUURGGGUUAGGRGGUTGUTTRGAGGGRGGRGRGGGUAAUTGUAGTTUURGRGGRGGUAGGGUUAGRGAUUATUURGGTGGRGGURGRGAGTTUTTUTTRGAUXGGUAUURGGGRGTUTTRGUUTATGTGUTUAATTAUTAURGUAURGGUAAGUTGUAUTGUUURGUAGARGTGTGRGGGURGUTUTTRGAGGAGGAGUTGGUUTTUTGGGGUATRGARGAGAURGARGTGGAGUUUTGUTGUT GGATGAUUT  69RGTATTAAUAGGTTUUUUTURGRGUAUAUTGAUATATTTUTTATUUUUUATAATGAATTUAGUUATATGGUATTUTTTUUUATRGAAGGUUATRGGGAATGGUTTTAGGAAGUTGATTTTUAAGUTTTAAGRGGUAGUAGGTGURGGUAGXGRGGGGAURGATRGATGGAGAGAAGGRGGGUAAGARGURGGGAAGRGUATTUUTUUTUAAURGAGTGUUAUAAURGUUUTUURGAAGTGUUURGGGGUTTRGAGUATUAUUTRGRGGTAATURGGGAGGGTGGAGGGATGR GGUTGGAU  70GTUUAGURGUATUUUTUUAUUUTUURGGATTAURGRGAGGTGATGUTRGAAGUUURGGGGUAUTTRGGGAGGGRGGTTGTGGUAUTRGGTTGAGGAGGAATGRGUTTUURGGRGTUTTGUURGUUTTUTUTUUATRGATRGGTUUURGXGUTGURGGUAUUTGUTGURGUTTAAAGUTTGAAAATUAGUTTUUTAAAGUUATTUURGATGGUUTTRGATGGGAAAGAATGUUATATGGUTGAATTUATTATGGGGGATAAGAAATATGTUAGTGTGRGRGGAGGGGAAUUTGTT AATARG  71GTUTUTGUTATUUAGGUUTATGAGAATUAAGUTTTTAURGTTUUUTTTGGGGUUTGGATAGTTTTUAAGUTGUUTTUTUUTUAUATUATGTGAGTAGAGURGGUUAAGAAAAUUAGGGAGAUATUTUUUUUUAGUUTGGUUUAGAUTTUXGGGUUUUAUUUUAGGAGAGAGUUTUUUAUTUTGGUUAGTGUUUTGUAAAGUUTTRGUUUUAUUURGUUUUAATTTUTUUTTATGUUTGGUTUATTUUTTAGGGGUTGAGTTGGGAGATAAGAUTTGGGGTTGUAGTTGUTG GGGATUATT 72 AATGATUUUUAGUAAUTGUAAUUUUAAGTUTTATUTUUUAAUTUAGUUUUTAAGGAATGAGUUAGGUATAAGGAGAAATTGGGGRGGGGTGGGGRGAAGGUTTTGUAGGGUAUTGGUUAGAGTGGGAGGUTUTUTUUTGGGGTGGGGUUXGGAAGTUTGGGUUAGGUTGGGGGGAGATGTUTUUUTGGTTTTUTTGGURGGUTUTAUTUAUATGATGTGAGGAGAAGGUAGUTTGAAAAUTATUUAGGUUUUAAAGGGAARGGTAAAAGUTTGATTUTUATAGGUUTGGAT AGUAGAGAU  73GRGURGGGGUUTARGAAGUUTGGGURGGGGGURGGGGGGRGGGGARGRGGGAUURGGGGARGRGGGGRGUTUAGUUAGGUUUUUTUUAGURGRGURGGGGURGTUURGAGURGRGRGUAUAAARGGATGGGURGGTGURGUUTGURGGGXGRGRGGGGGTRGGTGUUTTUTGRGTGGRGRGRGTGTUUURGGGTUTURGTGRGGURGGRGUATTGGUUTRGRGUTUTURGGAGGGGAUTGAGUAGGTGAAUAGGUURGGAGUUTGTRGTGGAGGGGUURGGGAAGGUU TRGUTGUAGAAG 74 UTTUTGUAGRGAGGUUTTUURGGGUUUUTUUARGAUAGGUTURGGGUUTGTTUAUUTGUTUAGTUUUUTURGGAGAGRGRGAGGUUAATGRGURGGURGUARGGAGAUURGGGGAUARGRGRGUUARGUAGAAGGUAURGAUUUURGRGXGUURGGUAGGRGGUAURGGUUUATURGTTTGTGRGRGRGGUTRGGGARGGUUURGGRGRGGUTGGAGGGGGUUTGGUTGAGRGUUURGRGTUUURGGGTUURGRGTUUURGUUUUURGGUUUURGGUUUAGGUTTRGT AGGUUURGGRGU 75 GUAUAAARGGATGGGURGGTGURGUUTGURGGGRGRGRGGGGGTRGGTGUUTTUTGRGTGGRGRGRGTGTUUURGGGTUTURGTGRGGURGGRGUATTGGUUTRGRGUTUTURGGAGGGGAUTGAGUAGGTGAAUAGGUURGGAGUUTGTXGTGGAGGGGUURGGGAAGGUUTRGUTGUAGAAGUAGGATGGGAGUAGGATURGUAGGGATGRGUAGRGGGGTURGRGGAGUTUURGGRGGGGGRGRGTTTUUAGGURGGGGAURGRGGTGUUAGUUUTGUUUTRGRGG ATRGGGTUTRG 76 RGAGAUURGATURGRGAGGGUAGGGUTGGUAURGRGGTUUURGGUUTGGAAARGRGUUUURGURGGGAGUTURGRGGAUUURGUTGRGUATUUUTGRGGATUUTGUTUUUATUUTGUTTUTGUAGRGAGGUUTTUURGGGUUUUTUUAXGAUAGGUTURGGGUUTGTTUAUUTGUTUAGTUUUUTURGGAGAGRGRGAGGUUAATGRGURGGURGUARGGAGAUURGGGGAUARGRGRGUUARGUAGAAGGUAURGAUUUURGRGRGUURGGUAGGRGGUAURGGUUU ATURGTTTGTGU 77 UTTRGGGUAGURGAGGGRGRGGGATGAUTRGGGUAGGRGTGTGGUUTURGAUTGAUUAGGGTTGGGUURGGGATUAGAGUTTTGUTTGGAAGTTUTUTGGGTGGUAGAGAUTRGGGUAAURGGAGUUTGAGUUTUURGRGGGGAAGAGUTXGGAUTURGGAGGTRGRGURGUUTTGGGUTTGAATTUAGTUUTTUUTTAUTAUTRGTGGUTTTGUTTAUATUATTTAAUTUTUUATTTUUTTGTUTATAUAGTGGGUTUATAAGGAGGRGGTAAGGAGATGATGATGTUTTTGA AGAGTT  78AAUTUTTUAAAGAUATUATUATUTUUTTAURGUUTUUTTATGAGUUUAUTGTATAGAUAAGGAAATGGAGAGTTAAATGATGTAAGUAAAGUUARGAGTAGTAAGGAAGGAUTGAATTUAAGUUUAAGGRGGRGRGAUUTURGGAGTUXGAGUTUTTUUURGRGGGAGGUTUAGGUTURGGTTGUURGAGTUTUTGUUAUUUAGAGAAUTTUUAAGUAAAGUTUTGATUURGGGUUUAAUUUTGGTUAGTRGGAGGUUAUARGUUTGUURGAGTUATUURGRGUUUTRGGU TGUURGAAG  79URGUAGUUAUTAUTRGUUUUAUUTTUTUAUUTURGAGGGGTTGAAUURGAGTTGTUTGTTGGGGTTTAGGGAUTUAGGATRGGGTUTGAGGGAUTTRGAUATAAGTGRGTGGRGGTAGTGGGTATAUAGGGGAGGGGGGURGGGUUUUTXGUTUUTTUTGGAAAURGGGUUUUAUTTGUAGGUURGGUUAUUTTGGGTTUTGGTGGURGAAGURGGAGUTGTGTTTUTRGUAGAUTRGGGGAGUTAUATTGTGRGTAGGUAATTGTTTAGTTTGAAAGGAGGUAUATTTUAUUA RGUAGU  80GUTGRGTGGTGAAATGTGUUTUUTTTUAAAUTAAAUAATTGUUTARGUAUAATGTAGUTUUURGAGTUTGRGAGAAAUAUAGUTURGGUTTRGGUUAUUAGAAUUUAAGGTGGURGGGUUTGUAAGTGGGGUURGGTTTUUAGAAGGAGXGAGGGGUURGGUUUUUUTUUUUTGTATAUUUAUTAURGUUARGUAUTTATGTRGAAGTUUUTUAGAUURGATUUTGAGTUUUTAAAUUUUAAUAGAUAAUTRGGGTTUAAUUUUTRGGAGGTGAGAAGGTGGGGRGAGTAG TGGUTGRGG  81UTUUUTGRGGUTUUAUTAGTTTTUTTRGUUURGUUUAGURGUUUAUTUTTUTRGGUTAGGGAAGAAGAUUAGAGGGTGUTUAGUTGGAAAAUTUTGGTGTUTUAGUTTAGGGUUTUUTURGGGAAGAGUTAAUTGUTUUUAGGTGAAGUXGGTGUURGRGGGRGGTURGTAUAUUURGUAGURGGUTRGUAURGUTRGAGAGUUTRGGURGUTGTGTUTTUUARGTUTGUAGUTUAGUUAGGGRGRGUAGGGRGAGTGGGGTUUAUTGGRGGGTAAAGGGGAUUAGGARGG RGAGGATGG  82UUATUUTRGURGTUUTGGTUUUUTTTAUURGUUAGTGGAUUUUAUTRGUUUTGRGRGUUUTGGUTGAGUTGUAGARGTGGAAGAUAUAGRGGURGAGGUTUTRGAGRGGTGRGAGURGGUTGRGGGGTGTARGGAURGUURGRGGGUAUXGGUTTUAUUTGGGAGUAGTTAGUTUTTUURGGAGGAGGUUUTAAGUTGAGAUAUUAGAGTTTTUUAGUTGAGUAUUUTUTGGTUTTUTTUUUTAGURGAGAAGAGTGGGRGGUTGGGRGGGGRGAAGAAAAUTAGTGGAG URGUAGGGAG 83 TAUATATGTAUARGTGTGUAUAURGGTUUTRGRGAGGRGGUUURGRGGGGTUATAGRGRGGUAGRGTAGGURGRGGGGTAGGGGTUUAGUTGGGGUTTGUUUATGUURGGUAGUAGGATGTAGGRGAGRGGRGGUTGUAGUUXGGGGUTGGGUUAXGRGUTGUAGTTGUARGGGATUATGTAGUURGGGTTGUURGGGUTGGGGTGRGAGTGRGTGTGUUUUURGGRGGRGGRGGURGURGUTGUAGURGURGURGURGRGURGTGGAAGGRGUURGRGUURGRGGTRGGG TAGUUUAGU  84GUTGGGUTAUURGAURGRGGGRGRGGGRGUUTTUUARGGRGRGGRGGRGGRGGUTGUAGRGGRGGURGURGURGURGGGGGGUAUARGUAUTRGUAUUUUAGUURGGGUAAUURGGGUTAUATGATUURGTGUAAUTGUAGRGXGTGGUUUAGUUUXGGGUTGUAGURGURGUTRGUUTAUATUUTGUTGURGGGUATGGGUAAGUUUUAGUTGGAUUUUTAUUURGRGGUUTARGUTGURGRGUTATGAUUURGRGGGGURGUUTRGRGAGGAURGGTGTGUAUARGT GTAUATATGTA 85 ARGURGUUAGUTUTGATTGGUUUAGRGGTAGGAAAGGTTAAAUUAAAAATTTTTTTAUAGUUUTAGTGTGRGUUTGTAGUTRGGAAAATTAATTGTGGUTATAGURGUUTRGATRGUTGTUTUUUUAGUUTRGURGRGGURGUTURGGGAXGRGUURGUURGURGUURGGUTUTUUUUUUUTTTGGGUTGUTGUTGUTGUTGUTGTGAUTGUTGUTGRGAGAGGAGGAGGAGGAGGAGGAAGUAGRGGGGGGGGGAGRGGGGGGTGGGGGGGGAGAUUAAGAAGTAUAGTT GGGAGRGAG  86UTRGUTUUUAAUTGTAUTTUTTGGTUTUUUUUUUUAUUUUURGUTUUUUUUUURGUTGUTTUUTUUTUUTUUTUUTUUTUTRGUAGUAGUAGTUAUAGUAGUAGUAGUAGUAGUUUAAAGGGGGGGAGAGURGGGRGGRGGGRGGGRGXGTUURGGAGRGGURGRGGRGAGGUTGGGGAGAUAGRGATRGAGGRGGUTATAGUUAUAATTAATTTTURGAGUTAUAGGRGUAUAUTAGGGUTGTAAAAAAATTTTTGGTTTAAUUTTTUUTAURGUTGGGUUAATUAGAG UTGGRGGRGT 87 AGGUAGAATAAAAUUUUUTUUUUTAGAGURGGGGTGGUTUAGRGGAATUATRGAGAATGAGAURGUTGGTTGUTAATGGGUTTGGGGAAAATGGGATGUAATTTUUURGGTGTTTTTUAGGUUUAGAGUTATTGAATAAATGAAGTGRGXGURGGRGGAGTUAGTAAUTUAUTGRGRGGUTUURGGUAGGRGGGGGRGGAGTGGGGGURGUAGAAURGGARGTGUUTGGRGAGGTTUAGAGGRGUTAGRGGTGTGGTGGGTGGGUAGRGURGGGATGGUTGGAGGGAGGG GUTARGGGGG 88 UUUURGTAGUUUUTUUUTUUAGUUATUURGGRGUTGUUUAUUUAUUAUAURGUTAGRGUUTUTGAAUUTRGUUAGGUARGTURGGTTUTGRGGUUUUUAUTURGUUUURGUUTGURGGGAGURGRGUAGTGAGTTAUTGAUTURGURGGXGRGUAUTTUATTTATTUAATAGUTUTGGGUUTGAAAAAUAURGGGGAAATTGUATUUUATTTTUUUUAAGUUUATTAGUAAUUAGRGGTUTUATTUTRGATGATTURGUTGAGUUAUUURGGUTUTAGGGGAGGGGGTTTTA TTUTGUUT  89UUUTGURGAGGTTATGGTGUUTGTTTUUAGUAGUTUTGAGUUUARGRGGAAUATGAGTRGUTGATAAAUAAGGUTTGUAGTTGRGAGGGGUUTRGGGGTGGGTTTUTGGTTGAGGAARGURGGGGAATGGUTGATGGAGGAAGUUUUTGUXGGGAGUAGGAGUAGRGGAAAUUTAUATRGGUUAUAUAGATUURGGRGGGGGAGGGAGGGUAARGUTUAGAAGUAGAGUTGRGRGATGTGUUTTTUTGGGUTGUTUAGUUUAGGGUURGAGAUUTGUTTGUUTGAGGAGG GGUAGATGUA 90 TGUATUTGUUUUTUUTUAGGUAAGUAGGTUTRGGGUUUTGGGUTGAGUAGUUUAGAAAGGUAUATRGRGUAGUTUTGUTTUTGAGRGTTGUUUTUUUTUUUURGURGGGATUTGTGTGGURGATGTAGGTTTURGUTGUTUUTGUTUUXGGUAGGGGUTTUUTUUATUAGUUATTUUURGGRGTTUUTUAAUUAGAAAUUUAUUURGAGGUUUUTRGUAAUTGUAAGUUTTGTTTATUAGRGAUTUATGTTURGRGTGGGUTUAGAGUTGUTGGAAAUAGGUAUUATAAUUT RGGUAGGG  91GGUUURGARGUUAGUTGTUUUUURGAGAAGUTURGUAGRGRGTRGUTUUAGGAGTGRGAGGAGUUATAGATGGRGUTGURGTUUAGUUAGUURGTUAUUTGGTTGGUUTGRGGGGUARGRGGRGGGTGAGUURGGGTRGAGAGGRGGRGGXGGGUUAGGGAAGGURGGGGUURGGRGGGTGTURGRGGUTGGGGAGTGGGRGUUTUTUUUUTUUAGGUUUTGUUAGGUURGAAGUUUAGGUUUUAUUTGGUTGGGGTGRGGTUUUTTUURGURGUUTTUUURGUUTUAU UAGGTUURGGG 92 UURGGGAUUTGGTGAGGRGGGGAAGGRGGRGGGAAGGGAURGUAUUUUAGUUAGGTGGGGUUTGGGUTTRGGGUUTGGUAGGGUUTGGAGGGGAGAGGRGUUUAUTUUUUAGURGRGGAUAUURGURGGGUUURGGUUTTUUUTGGUUXGURGURGUUTUTRGAUURGGGUTUAUURGURGRGTGUUURGUAGGUUAAUUAGGTGARGGGUTGGUTGGARGGUAGRGUUATUTATGGUTUUTRGUAUTUUTGGAGRGARGRGUTGRGGAGUTTUTRGGGGGGAUAGUT GGRGTRGGGGUU 93 GTTTTTRGTTTUUAATGUUUTGGAUUTTTAGAGAGUTUATUATUTTTUUTTATGGTRGUAAGTUTTGAAAGAAAGAGUAAGAGAGRGAGRGGGTTGGGTTGGGGUTGGAGTAGURGAGGURGGUUTGGGTURGGGUAGTUAGGUUTGAXGRGGUUURGRGUUUTTUUURGGUAGAGAAGUURGGGARGGUUATGTGRGTGGGUTGRGGGAGTUAGATUUARGAUUAGTTTATUUTGRGGGTGTRGUURGAUUTRGAGTGGUARGRGGUUTGUUTUAAGTGTGURGAGTGUAG UUAGTAUU  94GGTAUTGGUTGUAUTRGGUAUAUTTGAGGUAGGURGRGTGUUAUTRGAGGTRGGGRGAUAUURGUAGGATAAAUTGGTRGTGGATUTGAUTUURGUAGUUUARGUAUATGGURGTUURGGGUTTUTUTGURGGGGAAGGGRGRGGGGURGXGTUAGGUUTGAUTGUURGGAUUUAGGURGGUUTRGGUTAUTUUAGUUUUAAUUUAAUURGUTRGUTUTUTTGUTUTTTUTTTUAAGAUTTGRGAUUATAAGGAAAGATGATGAGUTUTUTAAAGGTUUAGGGUATTGGAA ARGAAAAAU  95TGTAUUUTGGGTTUUUUTAUTTTUUUUUAGTTGGGUUUUAUATUTGGGTUTGGUTUTTAAGTUTTGGGUATATGGGATAGAGAATTTAAGGATUUAGATAUTTTGUAATUUURGGGUTAGTAGGAGGAAAAGTGGUTUUTUTGGGAAUUXGGATGGGURGAAUAGUAGUTTURGGUAGAGGUUUTUAGGTTTUAGAGTTUTUUAAGTGGGGAGAAGGGRGGUUTGUTUAGUURGRGUAUURGTGGUAGUAUUAUAGAGGGAATAAAAGUAGGGTAAAUAATTTGAAGATUU UUARGAAGG  96UUTTRGTGGGGATUTTUAAATTGTTTAUUUTGUTTTTATTUUUTUTGTGGTGUTGUUARGGGTGRGRGGGUTGAGUAGGURGUUUTTUTUUUUAUTTGGAGAAUTUTGAAAUUTGAGGGUUTUTGURGGAAGUTGUTGTTRGGUUUATUXGGGTTUUUAGAGGAGUUAUTTTTUUTUUTAUTAGUURGGGGATTGUAAAGTATUTGGATUUTTAAATTUTUTATUUUATATGUUUAAGAUTTAAGAGUUAGAUUUAGATGTGGGGUUUAAUTGGGGGAAAGTAGGGGAAUUUA GGGTAUA  97UTGUUTGUAGUURGAGGAGUTGAUTAATGUTUTGGAGATUAGUAAUATRGTGTTUAUUAGUATGTTTGUUUTGGAGATGUTGUTGAAGUTGUTGGUUTGRGGUUUTUTGGGUTAUATURGGAAUURGTAUAAUATUTTRGARGGUATUATXGTGGTUATUAGGTGGGTUUUUAUUUTUTUUUUAGGAAGAGGGGUURGGGAAGUTUUAUTUTUTGGUAGAAATUUUAUUTGUAGAGUAAAAUUUAGAGUAUAGGAGGAAGTARGARGATAGUTUTTTATGAUAGGURGTGG GAAGUAGGT  98AUUTGUTTUUUARGGUUTGTUATAAAGAGUTATRGTRGTAUTTUUTUUTGTGUTUTGGGTTTTGUTUTGUAGGTGGGATTTUTGUUAGAGAGTGGAGUTTUURGGGUUUUTUTTUUTGGGGAGAGGGTGGGGAUUUAUUTGATGAUUAXGATGATGURGTRGAAGATGTTGTARGGGTTURGGATGTAGUUUAGAGGGURGUAGGUUAGUAGUTTUAGUAGUATUTUUAGGGUAAAUATGUTGGTGAAUARGATGTTGUTGATUTUUAGAGUATTAGTUAGUTUUTRGGGUTG UAGGUAG  99UUUUUTGGAAGGTGUTAATTTUTGAGGUUTUUAGGGGAAUAATGAGGAGGAGAAAAATUUUAGTGTGAAAATGATGTUAGRGGAGTTGUTTGTUUUTTTGTUTGTUTGTUUTTTATAAGUUATUUUUTUTGUAGAGGUTGUTGGGURGGXGGGAUTGTUUUTUUTUUTUTTTGUUUATUTUTTTGGURGGGTGAGTUTUATUTUAAGAUTUUTUAUTGTAUTUTUAUAGUTTGGTUTUUATAUAAATGAAGTTUAUUUTUTGGGUTUTGAAAUAGUTTUTUAUTUUAUUUAAA AAGATUU 100GGATUTTTTTGGGTGGAGTGAGAAGUTGTTTUAGAGUUUAGAGGGTGAAUTTUATTTGTATGGAGAUUAAGUTGTGAGAGTAUAGTGAGGAGTUTTGAGATGAGAUTUAUURGGUUAAAGAGATGGGUAAAGAGGAGGAGGGAUAGTUUXGURGGUUUAGUAGUUTUTGUAGAGGGGATGGUTTATAAAGGAUAGAUAGAUAAAGGGAUAAGUAAUTURGUTGAUATUATTTTUAUAUTGGGATTTTTUTUUTUUTUATTGTTUUUUTGGAGGUUTUAGAAATTAGUAUUT TUUAGGGGG101 RGTRGTAGTTGGGGURGAGGTURGTGTGGTAGGGGAUTGGGAUAGGAGTRGGAGTRGUAGGGRGAGGGUUUAGATRGGGGUTGGAAGUUAUAGGUUTUTUAUUTRGUTTGGURGAGRGGURGGGAUTURGGGUURGUAGRGGATGGAAGXGGGRGUTGUUUTGUURGGRGGRGGURGGGGUUTTUTGGGGTTTTUURGRGGGRGGUTRGGUAGGGRGGUAGUAGUTGURGRGGAGAGGARGAURGUUAURGGTGUAAUUAGGTUUURGRGUUUUTGUURGGUUTUTGGG GRGUUUAAGRG102 RGUTTGGGRGUUUUAGAGGURGGGUAGGGGRGRGGGGAUUTGGTTGUAURGGTGGRGGTRGTUUTUTURGRGGUAGUTGUTGURGUUUTGURGAGURGUURGRGGGAAAAUUUUAGAAGGUUURGGURGURGURGGGUAGGGUAGRGUUXGUTTUUATURGUTGRGGGUURGGAGTUURGGURGUTRGGUUAAGRGAGGTGAGAGGUUTGTGGUTTUUAGUUURGATUTGGGUUUTRGUUUTGRGAUTURGAUTUUTGTUUUAGTUUUUTAUUAUARGGAUUTRGGUUU UAAUTARGARG103 RGUAGGURGUAGTGARGTRGGRGGGRGUTGUATTAAGGRGAGGGGGUTTUUAGAGUTUAGUUAAUAGUUAGGAGUAGTGAUUAAGURGURGGAGUTGGGGAGAGARGUAURGGGGRGGRGAUTGGGUUAGGAGAUUAGGGAUTGAGGGAXGRGUURGGGGAGGGUUAGUAAUAAGURGRGGUURGGGRGRGAUAGRGGRGGTURGRGURGAGURGTTUUAGURGURGGUUTAUTGTAGURGUTGRGUUAGGAUATTTTTTTTTAAAGUTUTUUAAGUTGUUUUUUTUUT UUURGAUTUUT104 AGGAGTRGGGGAGGAGGGGGGUAGUTTGGAGAGUTTTAAAAAAAAATGTUUTGGRGUAGRGGUTAUAGTAGGURGGRGGUTGGAARGGUTRGGRGRGGAURGURGUTGTRGRGUURGGGURGRGGUTTGTTGUTGGUUUTUUURGGGRGXGTUUUTUAGTUUUTGGTUTUUTGGUUUAGTRGURGUUURGGTGRGTUTUTUUUUAGUTURGGRGGUTTGGTUAUTGUTUUTGGUTGTTGGUTGAGUTUTGGAAGUUUUUTRGUUTTAATGUAGRGUURGURGARGTUAUTGRG GUUTGRG 105RGUUUTRGGUUURGUUUUUTGUURGUUUUTUUUTUTRGGGGTURGGGGRGRGAGUTGRGGRGGRGGRGGUUARGGTUATTGGRGURGAGRGGTTURGGUTGAUTGGARGGGGRGGGRGTUURGGGUAGUUTAGRGRGGTAUUTUURGUUUXGRGRGUUUAGURGGRGAGGGAUATTGGAUUAGGGTRGGGGGTRGRGGURGUTUUAGRGAGGTAAGAGURGGGAAGAURGGGAGAGAUUAUUTUTTUUATUUTGGGGGGGTUUUTGGGGGARGGTUTUUUAURGGTGUTG GGURGRGGRG106 RGURGRGGUUUAGUAURGGTGGGAGAURGTUUUUUAGGGAUUUUUUUAGGATGGAAGAGGTGGTUTUTUURGGTUTTUURGGUTUTTAUUTRGUTGGAGRGGURGRGAUUUURGAUUUTGGTUUAATGTUUUTRGURGGUTGGGRGRGXGGGGRGGGAGGTAURGRGUTAGGUTGUURGGGARGUURGUUURGTUUAGTUAGURGGAAURGUTRGGRGUUAATGAURGTGGURGURGURGURGUAGUTRGRGUUURGGAUUURGAGAGGGAGGGGRGGGUAGGGGGRGG GGURGAGGGRG107 RGUUUTURGGUURGGUUTUUTURGURGGUURGGUTUURGAGAGGRGUAGUUAGUTUTURGUUATGTUTGRGGGGAUTUTURGAGGGGGRGGTUAGUATUUARGGGUTGAGURGGGGGTGGUURGURGRGUTUTUTGGUURGUTGAGTUUXGUAGUTUURGTTAUARGGUUTUURGGARGRGRGUTTUUATUTRGRGAUUURGGGGGRGUUTUUTURGAATAAGTATGTGGTGUUTGRGAGGAUUARGGTGGGAGUTGAGUAUTAUUUUAUUUURGAGGGGAUAGTGTGTGT ARGGGGARG 108RGTUUURGTAUAUAUAUTGTUUUUTRGGGGGTGGGGTAGTGUTUAGUTUUUAURGTGGTUUTRGUAGGUAUUAUATAUTTATTRGGAGGAGGRGUUUURGGGGTRGRGAGATGGAAGRGRGRGTURGGGAGGURGTGTAARGGGAGUTGXGGGAUTUAGRGGGUUAGAGAGRGRGGRGGGUUAUUUURGGUTUAGUURGTGGATGUTGAURGUUUUUTRGGAGAGTUUURGUAGAUATGGRGGAGAGUTGGUTGRGUUTUTRGGGAGURGGGURGGRGGAGGAGGURGGG URGGAGGGRG109 RGGGGTGUAGAGGTAAGAATGGGGGUAGAAAAUUUUUAGGTRGTGGTUUTGGGUUUAGAAAGTTGUUUUAGUUTGRGRGUUUUTTUUUAGUUUUTUAGGGTUUTTUUTUAUUURGRGGARGGTUUUATURGGGTGGUAAAGTTAGTGTGXGGRGUUTTGGAGUTUUUUUTURGGTUUUTUURGTUURGUURGTUTGUUUUTAGGTRGGUTAUUUUUUAAUUUUTURGUUTGTGUUAUUUTUTUUUUAGUUTTTGGTGGUAUTGUTUTUUTUUURGRGGGGUTRGGGUUTG GUTURGARGA110 TRGTRGGAGUUAGGUURGAGUUURGRGGGGAGGAGAGUAGTGUUAUUAAAGGUTGGGGAGAGGGTGGUAUAGGRGGAGGGGTTGGGGGGTAGURGAUUTAGGGGUAGARGGGRGGGARGGGAGGGAURGGAGGGGGAGUTUUAAGGRGUXGUAUAUTAAUTTTGUUAUURGGATGGGAURGTURGRGGGGTGAGGAAGGAUUUTGAGGGGUTGGGAAGGGGRGRGUAGGUTGGGGUAAUTTTUTGGGUUUAGGAUUARGAUUTGGGGGTTTTUTGUUUUUATTUTTAUUTUTGUAUUURG 111GGTAATTTRGGUUUUUTGAGUTTGGUTTAGTTTTTURGRGAAGTGUARGGGGGURGTTTTAGGATAUUUAGUTUUUAUUTGGAGUTUUAGAGUTUURGGGGAUUUUUTTGTURGUURGTUTUUTAGGGUUTGGUAUTUUUTGGUUUXGUAGUUXGGGGAUUTUUAUUTTUUUUAGGRGGUAGUUAUAGGTUURGURGGGUURGTURGAGGTUTGRGGURGURGAAGTRGGGGTUTUAGGGRGTUAGGGAGUAAUUAGGURGRGGGGAGGGAGGURGGRGURGGRGRGGAAT TTUTTTATU 112GATAAAGAAATTURGRGURGGRGURGGUUTUUUTUUURGRGGUUTGGTTGUTUUUTGARGUUUTGAGAUUURGAUTTRGGRGGURGUAGAUUTRGGARGGGUURGGRGGGAUUTGTGGUTGURGUUTGGGGAAGGTGGAGGTUUUXGGGUTGXGGGGUUAGGGAGTGUUAGGUUUTAGGAGARGGGRGGAUAAGGGGGTUUURGGGAGUTUTGGAGUTUUAGGTGGGAGUTGGGTATUUTAAAARGGUUUURGTGUAUTTRGRGGAAAAAUTAAGUUAAGUTUAGGGGG URGAAATTAUU113 AGATTGGGAATUTGGAGGGTAAAATGURGGGTUUUTTUURGAGUUUTTAGAGUURGAARGTTGTRGAATRGGTUAATGTUUAUUURGUTGUTUAUUTUTGTUUUAGUAGRGURGGGGUUAGRGRGUUUTUURGURGRGTUTGRGGAGUTGRGGGAAAAGUAGGTUUURGGGGGGTATRGAGTGTUUAGGGATATUUARGUAGAUATUUTTGTAUTTGUAAATAUAAAGAAAUAUAUAGGAUAGAGATUAGGUTRGRGUUAGGGUTRGATUTUUTRGGGGUTUAGGGTTGGA GGURGTAUA 114TGTARGGUUTUUAAUUUTGAGUUURGAGGAGATRGAGUUUTGGRGRGAGUUTGATUTUTGTUUTGTGTGTTTUTTTGTATTTGUAAGTAUAAGGATGTUTGRGTGGATATUUUTGGAUAUTRGATAUUUUURGGGGAUUTGUTTTTUURGUAGUTURGUAGARGRGGRGGGAGGGRGRGUTGGUUURGGRGUTGUTGGGAUAGAGGTGAGUAGRGGGGTGGAUATTGAURGATTRGAUAARGTTRGGGUTUTAAGGGUTRGGGAAGGGAUURGGUATTTTAUUUTUUAGATT UUUAATUT 115AGGGUUTUAGRGGRGGUAUURGUATUUTUTTTUTTATGGUAUAAGUTGAAGGRGGRGGURGRGGUAGGGAAGGTGTAGGGGTGRGGGAAGAGUURGURGUAGUURGGGAAUTTUTGUAGGARGUUUURGAARGAGUUUTTGUTGGGUAUXGGGATGAUTGGGTAGUTGRGRGGGUUTTTGGUUUUTGUUURGGUAUURGUUUURGRGUURGRGUUUARGUUUARGURGGUUAUAUAGUUAUUUUUAGRGURGUUUUUAUUAGTUUURGRGURGUURGAAGUAGUAGUUA UAGAGAGGUTG116 UAGUUTUTUTGTGGUTGUTGUTTRGGGRGGRGRGGGGAUTGGTGGGGGRGGRGUTGGGGGTGGUTGTGTGGURGGRGTGGGRGTGGGRGRGGGRGRGGGGGRGGGTGURGGGGUAGGGGUUAAAGGUURGRGUAGUTAUUUAGTUATUUXGGTGUUUAGUAAGGGUTRGTTRGGGGGRGTUUTGUAGAAGTTUURGGGUTGRGGRGGGUTUTTUURGUAUUUUTAUAUUTTUUUTGURGRGGURGURGUUTTUAGUTTGTGUUATAAGAAAGAGGATGRGGGTGURGURG UTGAGGUUUT117 AUTARGGTURGURGGGUUARGAUAAAATGUTUAGUUUUAAUTTRGARGRGUAUUAUAUTGUUATGUTGAUURGRGGTGAGUAAUAUUTGTUURGRGGUUTGGGUAUUUUAUUTGRGGUUATGATGTRGUAUUTGAARGGUUTGUAUUAUUXGGGUUAUAUTUAGTUTUARGGGURGGTGUTGGUAUUUAGTRGRGAGRGGUUAUUUTRGTUUTUATRGGGUTRGUAGGTGGUUARGTRGGGUUAGUTGGAAGAAATUAAUAUUAAAGAGGTGGUUUAGRGUATUAUAGR GGAGUTGAAGU118 GUTTUAGUTURGUTGTGATGRGUTGGGUUAUUTUTTTGGTGTTGATTTUTTUUAGUTGGUURGARGTGGUUAUUTGRGAGUURGATGAGGARGAGGGTGGURGUTRGRGAUTGGGTGUUAGUAURGGUURGTGAGAUTGAGTGTGGUUXGGGTGGTGUAGGURGTTUAGGTGRGAUATUATGGURGUAGGTGGGGTGUUUAGGURGRGGGAUAGGTGTTGUTUAURGRGGGTUAGUATGGUAGTGTGGTGRGRGTRGAAGTTGGGGUTGAGUATTTTGTRGTGGUURGGRGGA URGTAGT 119TGGGGTUAGTAGATUAGTUTUTTUAGAUAUTGATGUAGAAGUTGGGAUTGGTAAGTAGGTATTATGTGUTRGGAGRGUTAGGGGAUAGGAGUAAATGGAGAAGAAAAGRGGAGGUTTTUTURGUURGGAGTATRGATRGGAATUUURGUXGGTARGURGUAGAGGGUUUTRGURGTTGGGUUURGGGGGTTTAAUAAGUUUAGURGUTURGUAGGRGGUTRGGURGGAUTUTUAGAURGGTGUUTGGAAGAUAURGTUUUTGUUUUUUTUURGUUAAAUUTGUUTUTTUT UTTTUTUTUA120 TGAGAGAAAGAGAAGAGGUAGGTTTGGRGGGAGGGGGGUAGGGARGGTGTUTTUUAGGUAURGGTUTGAGAGTURGGURGAGURGUUTGRGGAGRGGUTGGGUTTGTTAAAUUUURGGGGUUUAARGGRGAGGGUUUTUTGRGGRGTAUXGGRGGGGATTURGATRGATAUTURGGGRGGAGAAAGUUTURGUTTTTUTTUTUUATTTGUTUUTGTUUUUTAGRGUTURGAGUAUATAATAUUTAUTTAUUAGTUUUAGUTTUTGUATUAGTGTUTGAAGAGAUTGATUTAU TGAUUUUA 121AGUTURGAGUUUARGUTGUAGUUAGATURGGATGAGTURGTUUTURGUUURGGGRGGGUTUTRGUTUTRGUTGGUUUTUAGRGURGRGUAGUUAGUAGUATUUUUAURGTGARGUTRGUATUAUAUURGGGRGURGGURGUUAUUATUXGRGURGURGURGTUAGGAUUUTUUTUURGGGUATRGTRGURGURGRGGGGTRGGGAGGARGRGGRGRGRGGGAGGRGGRGGTRGUAGGGRGAGUUURGGGARGUUURGAGURGGGGURGGGGURGGGGAGAGGGRGUAGR GAGGTGGGGGU122 GUUUUUAUUTRGUTGRGUUUTUTUUURGGUUURGGUUURGGUTRGGGGRGTUURGGGGUTRGUUUTGRGAURGURGUUTUURGRGRGURGRGTUUTUURGAUUURGRGGRGGRGARGATGUURGGGAGGAGGGTUUTGARGGRGGRGGRGXGGATGGTGGRGGURGGRGUURGGGTGTGATGRGAGRGTUARGGTGGGGATGUTGUTGGUTGRGRGGRGUTGAGGGUUAGRGAGAGRGAGAGUURGUURGGGGRGGAGGARGGAUTUATURGGATUTGGUTGUAGRGTGG GUTRGGAGUT123 AGUUUUTGGRGTUTTTUTRGGGGUUAGAGUTTTGUUTUTAAATGGTUTTTGTTTGUAGARGATUTGUTGTGUAGATUATGAAURGTRGURGUAGTGUUAGAUAGAUTUTTUATURGGAURGGURGTUUUAUTGGGTUUUUTUAGGAAAGXGUAGAAGURGGGGAUUUUAAAURGRGTUUAGUTUUTUUUAUURGUUUTTUTRGGAGURGRGUAUTTGGTAAUTTGRGTGTGAAGAAUTUARGGURGTGTTGUUAGGAAAUTGRGGGGAGUAGUUTTTGTGGTUTUAUATAT GRGTAAUAT 124ATGTTARGUATATGTGAGAUUAUAAAGGUTGUTUUURGUAGTTTUUTGGUAAUARGGURGTGAGTTUTTUAUARGUAAGTTAUUAAGTGRGRGGUTURGAGAAGGGRGGGTGGGAGGAGUTGGARGRGGTTTGGGGTUUURGGUTTUTGXGUTTTUUTGAGGGGAUUUAGTGGGARGGURGGTURGGATGAAGAGTUTGTUTGGUAUTGRGGRGARGGTTUATGATUTGUAUAGUAGATRGTUTGUAAAUAAAGAUUATTTAGAGGUAAAGUTUTGGUUURGAGAAAGARG UUAGGGGUT 125GUTTGAAUAGTTTTTTUUAATGTTRGATATTTAUATTTTTTGTUAUTTTTATTTTAGTUUTURGRGAAGGAGUUTUTUAAATATTUURGGAGUAUAGGUTAATTTAGAARGTGTTATUUTGAUAAGGRGGUUUAGUUAGGGAGGAGARGUXGGGGTURGRGURGRGGGGRGGGRGUTTUUURGGAUURGGGGGAGGRGGGARGUAGGRGAAGGURGUUURGGGAGAGRGGGGTUURGGGAGAGRGGGGTUURGGUTGTGGGGGARGRGGGURGAGGUTGTRGRGAAGURGUT GARGGURG 126RGGURGTUAGRGGUTTRGRGAUAGUUTRGGUURGRGTUUUUUAUAGURGGGAUUURGUTUTUURGGGAUUURGUTUTUURGGGGRGGUUTTRGUUTGRGTUURGUUTUUUURGGGTURGGGGAAGRGUURGUUURGRGGRGRGGAUUUXGGRGTUTUUTUUUTGGUTGGGURGUUTTGTUAGGATAAUARGTTUTAAATTAGUUTGTGUTURGGGAATATTTGAGAGGUTUUTTRGRGGAGGAUTAAAATAAAAGTGAUAAAAAATGTAAATATRGAAUATTGGAAAAAAU TGTTUAAGU 127UUTTRGGGGAUUAGAURGRGUTUUUTUAGATGGUTGGGGAGGGAGUAGAUTURGGGTGAAGGUTGGGGGAGGTAURGGGATGURGAGTARGUUAGAGUAGGRGGGGGATGGGTTURGGUTTUUTGURGUUTRGGUATUTRGUTTTGUAUXGGGUAAAGAAGGGGUUARGAURGGRGAAGAGRGRGTGGAGAUAUAGGGAUURGRGATUUAGGGGUAGGAAUUURGUUUUUTUURGGAAAUUTUURGGGUUUTGAGTRGTGUURGGRGUTUUUUAUUUUAUUUUAUUUU RGURGUUUUTGU128 GUAGGGGRGGRGGGGGTGGGGTGGGGTGGGGAGRGURGGGUARGAUTUAGGGUURGGGAGGTTTURGGGAGGGGGRGGGGTTUUTGUUUUTGGATRGRGGGTUUUTGTGTUTUUARGRGUTUTTRGURGGTRGTGGUUUUTTUTTTGUUXGGTGUAAAGRGAGATGURGAGGRGGUAGGAAGURGGAAUUUATUUUURGUUTGUTUTGGRGTAUTRGGUATUURGGTAUUTUUUUUAGUUTTUAUURGGAGTUTGUTUUUTUUUUAGUUATUTGAGGGAGRGRGGTUTGGT UUURGAAGG 129GGATGGGTUURGGRGRGGUUUAGUUUUTGUURGGUURGURGGGUAGAGAUTGAAURGRGGATUUUUAURGTUUTGTGGARGAURGGAUAGAGAGAGGUAUTGAURGATRGUUAGUAGUUTUURGGTGGGAURGRGTUTUUTGUAUAUUUXGRGUAGRGUUUUURGURGGAGURGUAURGGGUAAGURGGRGAGGGAGRGGGGUTGATTGGRGGURGURGGRGGUUAGGGGAGGGGGRGURGRGRGGGGUUATGGUAGGUTRGGAGGRGTUUTAGUURGAGURGGAGURG ATURGAGUUUA130 TGGGUTRGGATRGGUTURGGUTRGGGUTAGGARGUUTURGAGUUTGUUATGGUUURGRGRGGRGUUUUUTUUUUTGGURGURGGRGGURGUUAATUAGUUURGUTUUUTRGURGGUTTGUURGGTGRGGUTURGGRGGGGGGRGUTGRGXGGGGTGTGUAGGAGARGRGGTUUUAURGGGAGGUTGUTGGRGATRGGTUAGTGUUTUTUTUTGTURGGTRGTUUAUAGGARGGTGGGGATURGRGGTTUAGTUTUTGUURGGRGGGURGGGUAGGGGUTGGGURGRGURGG GAUUUATUU 131AGAAGAGGAAAAURGAGURGGRGGUTGRGGURGGGUATRGTATGGRGUTAGAGAAUTTRGGRGGRGAGRGGGAUUTGRGUUTGGGURGURGUUTUUURGUURGRGGTUURGGGAURGTTAUTTTGAAAAGGAGTUURGAGGUUTGGRGUUXGGRGRGRGATRGGGAUUURGRGTUUAGUTURGGGGAUURGGURGGRGUUUUUUAUURGRGAGRGGUURGRGAGUUAUTUTUAGGUUURGGGAAAUTTTUAAGAGGGTUTGGGGGGTGGGGGGAGTAAAAAGGGGAGG GGGTAGGUTGGG132 UUUAGUUTAUUUUUTUUUUTTTTTAUTUUUUUUAUUUUUUAGAUUUTUTTGAAAGTTTUURGGGGUUTGAGAGTGGUTRGRGGGURGUTRGRGGGTGGGGGGRGURGGURGGGTUUURGGAGUTGGARGRGGGGTUURGATRGRGRGUXGGGRGUUAGGUUTRGGGAUTUUTTTTUAAAGTAARGGTUURGGGAURGRGGGRGGGGAGGRGGRGGUUUAGGRGUAGGTUURGUTRGURGURGAAGTTUTUTAGRGUUATARGATGUURGGURGUAGURGURGGUTRGGTTT TUUTUTTUT133 TTUTUUUTGAAAUTTGTTTGAAAAUUUAAGTGAAAAURGRGARGATUTGUAUAUTUAAAAGUAAGTGUUAAGTAAGTGUUUTGRGGTGGUUTRGGRGRGUUURGGGGTGAGRGRGUAAAGURGGGAGRGAGGTGURGRGAGUTGRGRGUXGUUURGGGURGGUUTRGRGTGTGGGRGGUTGGGRGGUTGGGRGGRGGGAGGARGGUUTURGRGGGUTURGGGAUTGUURGGUTGGUTGUTRGUAUAAUTTTTTTTTTTTUUUURGTUTGUTGAUTTTTRGGGUUAGGTGAAGT GTTTGGG 134UUUAAAUAUTTUAUUTGGUURGAAAAGTUAGUAGARGGGGGAAAAAAAAAAAGTTGTGRGAGUAGUUAGURGGGUAGTUURGGAGUURGRGGAGGURGTUUTUURGURGUUUAGURGUUUAGURGUUUAUARGRGAGGURGGUURGGGGXGGRGRGUAGUTRGRGGUAUUTRGUTUURGGUTTTGRGRGUTUAUUURGGGGRGRGURGAGGUUAURGUAGGGUAUTTAUTTGGUAUTTGUTTTTGAGTGTGUAGATRGTRGRGGTTTTUAUTTGGGTTTTUAAAUAAGTT TUAGGGAGAA135 ATTURGGGAGATURGRGUUUAGGUURGRGRGUTRGGGGURGUTUTGGUUTUAGAGURGUTGUURGAUUUAGGAAURGGUAURGRGTRGUUAAGGGUAGTUATTGGRGGURGUAGARGGAGGAGGARGGRGTTGGURGGGARGRGGAUAGXGUAGGGUAGRGGRGGGGGRGRGGGURGGGGUUARGGGRGUAGGGGURGGAGURGUTGUAGURGUAAGURGTTGUARGTGGAUTTUAAGGAGUTRGGUTGGGARGAUTGGATUATRGRGURGUTGGAUTARGAGGRGTA UUAUTGRGAGGG136 UUUTRGUAGTGGTARGUUTRGTAGTUUAGRGGRGRGATGATUUAGTRGTUUUAGURGAGUTUUTTGAAGTUUARGTGUAARGGUTTGRGGUTGUAGRGGUTURGGUUUUTGRGUURGTGGUUURGGUURGRGUUUURGURGUTGUUUTGXGUTGTURGRGTUURGGUUAARGURGTUUTUUTURGTUTGRGGURGUUAATGAUTGUUUTTGGRGARGRGGTGURGGTTUUTGGGTRGGGUAGRGGUTUTGAGGUUAGAGRGGUUURGAGRGRGRGGGUUTGGGRGRGGATU TUURGGAAT 137GGGGURGUAGGGGGUTGGAAGGAAGTGRGUAGTGTGGRGAGAGRGRGAAUAAAGUUUTUTURGGAGUUURGTUAUUUUTRGGTGAUUUUAGGUURGUUURGUTGAGURGRGGGGUTUURGGGUUTRGUUTURGAGUAGGURGUAURGUUXGGTGGGRGUARGTUAGGUUTURGRGGURGUURGGGUUAGTGUTUUUUTRGGTUUURGUAGGURGAGUURGRGGURGGUUURGAAGGRGRGAGGGAUAGRGRGGURGGRGGTGGAGUUTUAUTUAGGUARGGAGUUUAU AGAGRGAGGUTG138 UAGUUTRGUTUTGTGGGUTURGTGUUTGAGTGAGGUTUUAURGURGGURGRGUTGTUUUTRGRGUUTTRGGGGURGGURGRGGGUTRGGUUTGRGGGGAURGAGGGGAGUAUTGGUURGGGRGGURGRGGAGGUUTGARGTGRGUUUAUXGGGRGGTGRGGUUTGUTRGGAGGRGAGGUURGGGAGUUURGRGGUTUAGRGGGGRGGGUUTGGGGTUAURGAGGGGTGARGGGGUTURGGAGAGGGUTTTGTTRGRGUTUTRGUUAUAUTGRGUAUTTUUTTUUAGUUUU UTGRGGUUUU139 URGUTGUUUURGGAUUUTUTUTGUUTGUAUAAUTRGTRGUTUTTRGRGUTGUAGAAUUTGUAGUUUTGGGURGAGGAUAAUAAAGTGGUTTUAGTGTURGGGUTRGUUTRGGTGGTGTGAGRGARGUURGTURGATRGGRGTGGAGRGUXGGGUURGGAGRGGTGGAGRGRGRGGUTGUUTGRGTUUATGGTUTAGTGGUAGURGGGRGRGTGAGGAGRGGUAGGUUTTGAGGUTGTRGTRGAGGGUTUUTUUAUUAURGGURGGUTUUUAAGUUAGRGTTGRGUAGATGU ARGGUUAGU 140GUTGGURGTGUATUTGRGUAARGUTGGUTTGGGAGURGGURGGTGGTGGAGGAGUUUTRGARGAUAGUUTUAAGGUUTGURGUTUUTUARGRGUURGGUTGUUAUTAGAUUATGGARGUAGGUAGURGRGRGUTUUAURGUTURGGGUUXGGRGUTUUARGURGATRGGARGGGRGTRGUTUAUAUUAURGAGGRGAGUURGGAUAUTGAAGUUAUTTTGTTGTUUTRGGUUUAGGGUTGUAGGTTUTGUAGRGRGAAGAGRGARGAGTTGTGUAGGUAGAGAGGGTURG GGGGUAGRGG141 AUUATTGTUATAGTAAUAUAUAATTRGGGUUUARGTAGAUTTAATUURGAGAGGUAATTGTTUUUTTGUTTGGGRGGUTARGUTUUURGRGGGGUTGUUTGUAGUUUURGGGUUUTTGUAGUTURGGGARGGRGRGRGGRGGRGGAGGUXGRGGGGUUUUAAGRGUUAGUUTGGUUURGGRGUAGATGRGUTGUUURGGURGUAGUUUAGURGURGRGTGTGTTGTUAGGARGATRGGAAARGRGTGTGTGGGGAGATGGGTGUUAGTGTRGUUTTGTUUATTATUUAAAA UUUAGTRGU142 GRGAUTGGGTTTTGGATAATGGAUAAGGRGAUAUTGGUAUUUATUTUUUUAUAUARGRGTTTURGATRGTUUTGAUAAUAUARGRGGRGGUTGGGUTGRGGURGGGGUAGRGUATUTGRGURGGGGUUAGGUTGGRGUTTGGGGUUURGXGGUUTURGURGURGRGRGURGTUURGGAGUTGUAAGGGUURGGGGGUTGUAGGUAGUUURGRGGGGAGRGTAGURGUUUAAGUAAGGGAAUAATTGUUTUTRGGGATTAAGTUTARGTGGGUURGAATTGTGTGTTAUTA TGAUAATGGT143 RGRGTURGUUTUUUTGGAUUATTTUAUTUUTTUAAGTUAUTGGUTUUAURGRGTTTTUTGTTUUUAUUURGTUAUAAUTTTGGGGUTTAAAGGUAUUAGGATUTGGUATUUURGGGATGUUAUUTGTUTTUUAGGATGTGTUUTGGUTUUXGATGUUUUAAGUTAAGUUURGGAUAGGUTGGGAAUUTUTUUUTAGUAGUUUUTGUUUTGGTUUUTAGGGUUURGUAGGUUTTGGGGTGGUAGTGGUUTTGTUUUATGTUATUUUAGAGGUUTRGGGAGGUAGATGGTTTT GTUTAGGAA 144TTUUTAGAUAAAAUUATUTGUUTUURGAGGUUTUTGGGATGAUATGGGAUAAGGUUAUTGUUAUUUUAAGGUUTGRGGGGUUUTAGGGAUUAGGGUAGGGGUTGUTAGGGAGAGGTTUUUAGUUTGTURGGGGUTTAGUTTGGGGUATXGGGAGUUAGGAUAUATUUTGGAAGAUAGGTGGUATUURGGGGATGUUAGATUUTGGTGUUTTTAAGUUUUAAAGTTGTGARGGGGTGGGAAUAGAAAARGRGGTGGAGUUAGTGAUTTGAAGGAGTGAAATGGTUUAGGG AGGRGGARGRG145 UTTTUTTTGTTATUTUURGTGAAAUUTTUAUTTAGUAGGTGGARGGAGUUURGRGAURGGGUAGAGTURGGGUTRGUURGAGGAUAGGAGGAGGAGRGGGAGUURGRGRGTUURGGGAGAGRGUUURGAGTGUAGGTUUURGUUURGUUXGGRGAGUUURGUTGGAGRGAGUUUAGRGRGURGGGGUTGGGGGGRGGUUARGAUUUUUUUTGAAGGGGGTGGUUARGGAGRGUAUUURGAGAAGRGAGUUUUUUTUUUUAGAGRGUTGUTUUTGRGGUTGUTGUTGUT GUTGGTGAUUAA146 TTGGTUAUUAGUAGUAGUAGUAGURGUAGGAGUAGRGUTUTGGGGAGGGGGGUTRGUTTUTRGGGGTGRGUTURGTGGUUAUUUUUTTUAGGGGGGGTRGTGGURGUUUUUUAGUUURGGRGRGUTGGGUTRGUTUUAGRGGGGUTRGUXGGGRGGGGRGGGGAUUTGUAUTRGGGGRGUTUTUURGGGARGRGRGGGUTUURGUTUUTUUTUUTGTUUTRGGGRGAGUURGGAUTUTGUURGGTRGRGGGGUTURGTUUAUUTGUTAAGTGAAGGTTTUARGGGAGATA AUAAAGAAAG147 GUUAGRGRGGGURGURGGRGATGARGGURGRGAAGUAGGAGURGUAGUUUAUUURGGGGGUUAGGGRGAGUUAGGRGUAGURGGRGGAUUAGGTGAGAGTRGGUAGURGRGGUUAGGUUUTUURGGGAGGGGTGGUTUUAGTGRGRGUTUXGUURGUUTUURGUTTUUUAGGUTGGGUTUURGRGUUTUUUTUTTUTUAUUUTUUUURGUUURGUUUUAGTTUUAGGUTUTUUTGUTTUTUUARGGAUTUTGRGGGAAGTTAGAGUUTUTGRGTGRGUTURGGGGUURG GRGAGAGGATG148 UATUUTUTRGURGGGUUURGGAGRGUARGUAGAGGUTUTAAUTTUURGUAGAGTURGTGGAGAAGUAGGAGAGUUTGGAAUTGGGGRGGGGRGGGGGAGGGTGAGAAGAGGGAGGRGRGGGAGUUUAGUUTGGGAAGRGGGAGGRGGGXGGAGRGRGUAUTGGAGUUAUUUUTUURGGGAGGGUUTGGURGRGGUTGURGAUTUTUAUUTGGTURGURGGUTGRGUUTGGUTRGUUUTGGUUUURGGGGTGGGUTGRGGUTUUTGUTTRGRGGURGTUATRGURGGRG GUURGRGUTGGU149 GGUTGAAGGTGUUURGGGGAAUUURGGRGGGRGGUUUAURGAGGGAGGGAGAGGRGGURGGGAUUAAGGAATGGGGUUTUTTGGTTUUUUATTAARGUARGUTGAAGAAATUTGUTGRGUTUUTGARGGURGUTUAURGGGTTRGAGUUUXGTUUTUUTATAGURGGGGRGUTRGUTGGUUAAAGRGAUURGAGUAGGRGAATGAUUTTTAGGRGGARGGGGTTTTUUUTUTGUTTTUTTGTTTUTTTTGAGGAGARGGGTGTGTGTTTGTGAGGTGGGGATGGGGGAAGAG TGTUUUAG150 UTGGGAUAUTUTTUUUUUATUUUUAUUTUAUAAAUAUAUAUURGTUTUUTUAAAAGAAAUAAGAAAGUAGAGGGAAAAUUURGTURGUUTAAAGGTUATTRGUUTGUTRGGGTRGUTTTGGUUAGRGAGRGUUURGGUTATAGGAGGAXGGGGUTRGAAUURGGTGAGRGGURGTUAGGAGRGUAGUAGATTTUTTUAGRGTGRGTTAATGGGGAAUUAAGAGGUUUUATTUUTTGGTUURGGURGUUTUTUUUTUUUTRGGTGGGURGUURGURGGGGTTUUURGGGGU AUUTTUAGUU151 GRGAGTTGTAAAUUTUAGAGAAAGGUAUTTGTUUUUAGUAAAARGUTTGGAGAGGAURGTGUARGUTGTGUTGUUUURGUUURGAGARGRGURGGGURGURGGGTUAURGGTTTTURGAAAGGGAUURGGUAGAGAUAAAGTGUUTTRGUXGUTGRGATAGGTTGGTTTTAUTTTGUAATAAAUAGUUUUTAATGGGAURGGGRGURGGGRGGAGAGUTRGGUURGGGGRGRGGUUTTTGURGUUTGGUTUTGRGGGURGUUURGURGGGRGUUAGGTTTTGGGGGGTGG UURGGUUURG152 RGGGGURGGGUUAUUUUUUAAAAUUTGGRGUURGGRGGGGRGGUURGUAGAGUUAGGRGGUAAAGGURGRGUUURGGGURGAGUTUTURGUURGGRGUURGGTUUUATTAGGGGUTGTTTATTGUAAAGTAAAAUUAAUUTATRGUAGXGGRGAAGGUAUTTTGTUTUTGURGGGTUUUTTTRGGAAAAURGGTGAUURGGRGGUURGGRGRGTUTRGGGGRGGGGGUAGUAUAGRGTGUARGGTUUTUTUUAAGRGTTTTGUTGGGGAUAAGTGUUTTTUTUTGAGGTTT AUAAUTRGU153 TTGRGGAGTURGURGRGGUUTRGGUTUURGUURGGRGUURGGUUTGGUUUUAURGURGUTUAUTRGGTURGUATRGURGUUAUUTURGGAGUTGGTGGGGAGUURGGRGAGGGAGGGUUTGGAAGGGGUUUTGGGRGURGAGTUUUURGUXGGRGURGAUAURGUUTGUUAGGAGUAGURGGUUTGUTRGAGGTGAUTGUAGURGRGRGGTTUTGGRGRGGUTTUTTUAUUAAUATGAGRGGGTUTUTGRGTURGAGAGTGAGRGUAGURGGGUAGRGUUTAGTGGATT AUAGUAGARGU154 GRGTUTGUTGTAATUUAUTAGGRGUTGUURGGUTGRGUTUAUTUTRGGARGUAGAGAUURGUTUATGTTGGTGAAGAAGURGRGUUAGAAURGRGRGGUTGUAGTUAUUTRGAGUAGGURGGUTGUTUUTGGUAGGRGGTGTRGGRGUXGGRGGGGGAUTRGGRGUUUAGGGUUUUTTUUAGGUUUTUUUTRGURGGGUTUUUUAUUAGUTURGGAGGTGGRGGRGATGRGGAURGAGTGAGRGGRGGTGGGGUUAGGURGGGRGURGGGRGGGAGURGAGGURGRGGR GGAUTURGUAA155 TAGGAGGUURGUTUTGUAUUTTUUTTAUTGTGGARGGGUUUTUTGAGUTUTGAGGUUTGGRGGGAGAGRGRGTGUAGUTRGAGUTUUTUTTUUUAUUUAGUUUURGRGUUUTAUUTGGTRGAUUTTURGUAGUUTUAGUURGGTUTTGUXGRGUTRGTUUTTGUAUAGGTGGATUTRGRGUAUUURGGGUTTGATUTUAGUTRGURGUARGUUUAGGUTGTAUURGGTTAURGGTGUUAUUATUTGGURGGGUURGGGGUURGAGAURGUTGTUTGUAGAUAUUAGGGGGU AGGGGGTUA 156TGAUUUUUTGUUUUUTGGTGTUTGUAGAUAGRGGTUTRGGGUUURGGGUURGGUUAGATGGTGGUAURGGTAAURGGGTAUAGUUTGGGRGTGRGGRGAGUTGAGATUAAGUURGGGGTGRGRGAGATUUAUUTGTGUAAGGARGAGRGXGGUAAGAURGGGUTGAGGUTGRGGAAGGTRGAUUAGGTAGGGRGRGGGGGUTGGGTGGGAAGAGGAGUTRGAGUTGUARGRGUTUTUURGUUAGGUUTUAGAGUTUAGAGGGUURGTUUAUAGTAAGGAAGGTGUAGAGR GGGUUTUUTA157 UUTGTURGRGUTUUUAGUUUUUARGUAGUUAGAUAUURGRGGGTGUTGAGATGAAGAGTGAAGTUAUUAGGAGAGAUAGGAAAUAGRGRGGGUUTRGGGGAUURGAGGGRGRGGAAGUTRGGGGGURGGGGTRGAGUAUAGATTGGGAGGXGUAGRGUTGGGRGUARGTUUURGTRGGUURGGRGGUUUUTAATGAGGRGRGUTGTGRGUAGAUTGUTAAGAGUAGUATGAGUARGGUTURGGRGGUUUUUAGGGUUURGRGGGTRGGRGUUAUUURGGAGRGRGGTG UURGGAATUAUT158 AGTGATTURGGGUAURGRGUTURGGGGTGGRGURGAUURGRGGGGUUUTGGGGGURGURGGAGURGTGUTUATGUTGUTUTTAGUAGTUTGRGUAUAGRGRGUUTUATTAGGGGURGURGGGURGARGGGGARGTGRGUUUAGRGUTGXGUUTUUUAATUTGTGUTRGAUUURGGUUUURGAGUTTURGRGUUUTRGGGTUUURGAGGUURGRGUTGTTTUUTGTUTUTUUTGGTGAUTTUAUTUTTUATUTUAGUAUURGRGGGTGTUTGGUTGRGTGGGGGUTGGGAGRGR GGAUAGG 159UATUUTTUURGGAAAAGTAUAAAUAGTTUUTUAGARGAGGTUUUUUAUUTUUUARGRGUTUUUUAGUUUTUUUTUUUTGRGGAGAGUUURGRGAUAGUUTUUUUAAUAUUTGTGAATUATURGGGAGGUTGUUAURGURGAGRGATURGXGUAUUAUUUUUTTUURGGGUURGGGUARGGUUAGGGAGGAUAGTTAGGGTTGTTGUTTTATAATTATUAUTTTTAATUTUTAATARGAUUAGUAUAAGTAGUUTTTGTUTUUURGUUUTGATTTGAGUATURGAGGGUUUUU RGAGGUAG 160UTGUUTRGGGGGGUUUTRGGATGUTUAAATUAGGGRGGGGAGAUAAAGGUTAUTTGTGUTGGTRGTATTAGAGATTAAAAGTGATAATTATAAAGUAAUAAUUUTAAUTGTUUTUUUTGGURGTGUURGGGUURGGGAAGGGGGTGGTGXGRGGATRGUTRGGRGGTGGUAGUUTUURGGATGATTUAUAGGTGTTGGGGAGGUTGTRGRGGGGUTUTURGUAGGGAGGGAGGGUTGGGGAGRGRGTGGGAGGTGGGGGAUUTRGTUTGAGGAAUTGTTTGTAUTTTTURGG GAAGGATG 161URGGAGARGAUTUUAGGGUTGGGTGAGGRGUTGAUUUUUAGGAGUTGGGGTGURGAGGGGRGRGGGUUAUAGRGGTGRGAGUUAGTRGGGRGUURGRGRGGTGGGGRGRGRGGRGRGGGGATTGGRGGGRGUTUUURGGTGUUXGUAGUTUTTUAGXGTAGURGGGAAGAGURGRGRGTUTGRGRGUUAGUUUURGUUUTGGGUURGURGUURGAGUTUTUTGGRGUAGRGUTAGUTURGURGRGUTUAGUTGUUUTGRGURGGUAUUUUTGGTUATGAGRGUUUUUTRG ARGUTGUUUU162 GGGGUAGRGTRGAGGGGGRGUTUATGAUUAGGGGTGURGGRGUAGGGUAGUTGAGRGRGGRGGAGUTAGRGUTGRGUUAGAGAGUTRGGGRGGRGGGUUUAGGGRGGGGGUTGGRGRGUAGARGRGRGGUTUTTUURGGUTAXGUTGAAGAGUTGXGGGUAURGGGGAGRGUURGUUAATUUURGRGURGRGRGUUUUAURGRGRGGGRGUURGAUTGGUTRGUAURGUTGTGGUURGRGUUUUTRGGUAUUUUAGUTUUTGGGGGTUAGRGUUTUAUUUAGUUUTGGA GTRGTUTURGG163 RGTGUUARGUAAARGURGTRGTURGUAURGGTRGRGAUTRGGUAAGGGAGRGGGRGGAGAGUTGAUTRGRGGRGGAGGGGGGTUAUTRGURGGARGGAARGUTGRGRGUAGUUATRGUTRGUTGTUAGURGRGTUUUAAUURGUTAGGAXGUTGGGUUUTGURGGRGGGATUUTUUUUTTAUTRGGAAAGGGGAGGRGURGGUUAUAGTAGGRGARGAGURGRGUTRGGUUAUUAURGRGGTGGURGGRGGAAUUUTRGUTUTUUUUURGTUAUUURGTRGAGGGGGAAG UTGAGGAGGG164 UUUTUUTUAGUTTUUUUUTRGARGGGGTGARGGGGGGAGAGRGAGGGTTURGURGGUUAURGRGGTGGTGGURGAGRGRGGUTRGTRGUUTAUTGTGGURGGRGUUTUUUUTTTURGAGTAAGGGGAGGATUURGURGGUAGGGUUUAGXGTUUTAGRGGGTTGGGARGRGGUTGAUAGRGAGRGATGGUTGRGRGUAGRGTTURGTURGGRGAGTGAUUUUUUTURGURGRGAGTUAGUTUTURGUURGUTUUUTTGURGAGTRGRGAURGGTGRGGARGARGGRGTTTGR GTGGUARG 165UURGGRGAGARGAATGUAGUAUARGGURGUUTTTATGGGGUURGUAGAUAGRGRGTRGUUAGGUTAAUUUTGRGTGGAAAATTRGGAGGTGGAAGGRGAGGRGUUTTATTGAGGGGGURGGUAGRGGRGGRGGRGGRGGRGAGGGGGRGGXGGGGGUTGTGRGGUURGGGURGGAAARGTGAGURGGGUTGGGGGRGGRGAUUAUUUURGRGUATTUUURGGUUUTUURGGGAUUAURGRGRGUUURGGURGGUAGGTGGAGGAGUAGGAGGRGUUUTUAURGTUUUR GUUUTGGGAGGA166 TUUTUUUAGGGRGGGGARGGTGAGGGRGUUTUUTGUTUUTUUAUUTGURGGURGGGGRGRGRGGTGGTUURGGGAGGGURGGGGAATGRGRGGGGGTGGTRGURGUUUUUAGUURGGUTUARGTTTURGGUURGGGURGUAUAGUUUUXGURGUUUUUTRGURGURGURGURGURGUTGURGGUUUUUTUAATAAGGRGUUTRGUUTTUUAUUTURGAATTTTUUARGUAGGGTTAGUUTGGRGARGRGUTGTUTGRGGGUUUUATAAAGGRGGURGTGTGUTGUATTRGT UTRGURGGG167 TUTGATGGTGGURGRGTGTTGGGUTTUAGTAGUUUUTGGAGUUUATUTAUTRGAAATAAATATTTUTRGGTAGUUATGGAAGTTGGAGUTGAGAAGUURGGGRGGGGGTGUUTGURGGATGUUUURGGTTUUUUTUTTTTUUUAUTRGUXGUUAAUTTAAUTTTTURGGGGGATGGATRGGUTGGTTGAGAAAGAGGAUARGUATAUTRGGTGTGAARGAGGTUARGAAUUUTUTGAUUUUAGGGAGAGATTTUTGUUAAGRGTAAGGTGTUTTTGGATUUUUUUAUUTUTGT UUUAGGT 168AUUTGGGAUAGAGGTGGGGGGATUUAAAGAUAUUTTARGUTTGGUAGAAATUTUTUUUTGGGGTUAGAGGGTTRGTGAUUTRGTTUAUAURGAGTATGRGTGTUUTUTTTUTUAAUUAGURGATUUATUUUURGGAAAAGTTAAGTTGGXGGRGAGTGGGAAAAGAGGGGAAURGGGGGUATURGGUAGGUAUUUURGUURGGGUTTUTUAGUTUUAAUTTUUATGGUTAURGAGAAATATTTATTTRGAGTAGATGGGUTUUAGGGGUTAUTGAAGUUUAAUARGRGGUU AUUATUAGA 169GGRGGRGATRGGAGRGGRGGRGGTGGTUTRGGRGGRGGRGGRGGRGGRGGRGGUAGGGAGRGGGUTUURGGTGURGGGUAURGGGRGGGRGGRGGGGAAGATGAURGRGGGRGURGGRGTGUTUUTTUTGUTGUTUTRGUTUTURGGRGXGUTURGGGTAAGTTGURGUUTUURGUUUURGURGTTRGGAAGUUURGGGUAGRGGGAGGTRGTUUURGGATUURGRGGGGRGUTUAUAUAUURGGRGGGGUTUTUURGGGUTUUUURGURGRGUTUUURGUTGUATUUAGU URGGRGUUU 170GGGRGURGGGUTGGATGUAGRGGGGAGRGRGGRGGGGGAGUURGGGAGAGUUURGURGGGTGTGTGAGRGUUURGRGGGATURGGGGARGAUUTUURGUTGUURGGGGUTTURGAARGGRGGGGGRGGGAGGRGGUAAUTTAUURGGAGXGRGURGGAGAGRGAGAGUAGUAGAAGGAGUARGURGGRGUURGRGGTUATUTTUUURGURGUURGUURGGTGUURGGUAURGGGAGUURGUTUUUTGURGURGURGURGURGURGURGURGAGAUUAURGURGURGUT URGATRGURGUU171 URGRGUTGRGGGTGGGRGGRGGGGTAGGAUTGRGGGGUAUTUTUAUUUURGAGUUUTRGGRGGGGUTGGAGUUTGRGTGTURGGRGGGGUUURGGGGGUTGGGAGUTGGGURGGGUTTGGGURGGGAAGURGGARGURGGGRGGGRGRGGXGRGGGAGGGRGUUURGGGAGGGRGAGRGGGTUTUURGUTGUUTUTGUAGAGUUURGRGGGURGGATTGUUAGUTTTGUTUTGRGUUTGGRGAGGTGRGRGGUURGRGGGGGUAGAGAGRGRGGRGGUTURGGGGGRGUU UUTGGGRGGA172 TURGUUUAGGGGRGUUUURGGAGURGURGRGUTUTUTGUUUURGRGGGURGRGUAUUTRGUUAGGRGUAGAGUAAAGUTGGUAATURGGUURGRGGGGUTUTGUAGAGGUAGRGGGAGAUURGUTRGUUUTUURGGGGRGUUUTUURGXGURGRGUURGUURGGRGTURGGUTTUURGGUUUAAGUURGGUUUAGUTUUUAGUUUURGGGGUUURGURGGAUARGUAGGUTUUAGUUURGURGAGGGUTRGGGGGTGAGAGTGUUURGUAGTUUTAUUURGURGUUUA UURGUAGRGRGG173 UURGGRGTURGRGTUUUURGGRGRGRGUUTTGGGGAURGGGTTGGTGGRGUUURGRGTGGGGUURGGTGGGUTTUURGGAGGGTTURGGGGGTRGGUUTGRGGRGRGTGRGGGGGAGGAGARGGTTURGGGGGAURGGURGRGAUTGRGGXGGRGGTGGTGGGGGGAGURGRGGGGATRGURGAGGGURGGTRGGURGUUURGGGTGURGRGRGGTGURGURGGRGGRGGTGAGGUUURGRGRGTGTGTUURGGUTGRGGTRGGURGRGUTRGAGGGGTUUURGTGGRGT UUUUTTUUUU174 GGGGAAGGGGARGUUARGGGGAUUUUTRGAGRGRGGURGAURGUAGURGGGAUAUARGRGRGGGGUUTUAURGURGURGGRGGUAURGRGRGGUAUURGGGGRGGURGAURGGUUUTRGGRGATUUURGRGGUTUUUUUUAUUAURGUXGURGUAGTRGRGGURGGTUUUURGGAAURGTUTUUTUUUURGUARGRGURGUAGGURGAUUUURGGAAUUUTURGGGAAGUUUAURGGGUUUUARGRGGGGRGUUAUUAAUURGGTUUUUAAGGRGRGRGURGGGGG ARGRGGARGURGGG175 UUTTTUTRGRGUUTTUUURGTRGUUURGGUUTRGUURGTGGTUTUTRGTUTTUTUURGGUURGUTUTTURGAAURGGGTRGGRGRGTUUUURGGGTGRGUUTRGUTTUURGGGUUTGURGRGGUUUTTUUURGAGGRGTURGTUURGGGXGTRGGRGTRGGGGAGAGUURGTUUTUUURGRGTGGRGTRGUUURGTTRGGRGRGRGRGTGRGUURGAGRGRGGUURGGTGGTUUUTUURGGAUAGGRGTTRGTGRGARGTGTGGRGTGGGTRGAUUTURGUUTTGURGGTRGU TRGUUUT 176AGGGRGAGRGAURGGUAAGGRGGAGGTRGAUUUARGUUAUARGTRGUARGAARGUUTGTURGGGAGGGAUUAURGGGURGRGUTRGGGRGUARGRGRGRGURGAARGGGGRGARGUUARGRGGGGAGGARGGGUTUTUUURGARGURGAXGUURGGGARGGARGUUTRGGGGAAGGGURGRGGUAGGUURGGGAAGRGAGGRGUAUURGGGGGARGRGURGAUURGGTTRGGAAGAGRGGGURGGGAGAAGARGAGAGAUUARGGGRGAGGURGGGGRGARGGGGAA GGRGRGAGAAAGG177 GGGRGGGTGGTTGGGGRGTURGGTTRGURGRGUUURGUUURGGUUUUAURGGTUURGGURGURGUUUURGRGUURGUTRGUTUUUTUURGTURGUURGTURGRGGUURGTURGTURGTURGTURGTRGTUUTUUTRGUTTGRGGGGRGUXGGGUURGTUUTRGRGAGGUUUUURGGURGGURGTURGGURGRGTRGGGGUUTRGURGRGUTUTAUUTAUUTAUUTGGTTGATUUTGUUAGTAGUATATGUTTGTUTUAAAGATTAAGUUATGUATGTUTAAGTARGUARGGUR GGTAUAG 178UTGTAURGGURGTGRGTAUTTAGAUATGUATGGUTTAATUTTTGAGAUAAGUATATGUTAUTGGUAGGATUAAUUAGGTAGGTAGGTAGAGRGRGGRGAGGUUURGARGRGGURGGARGGURGGURGGGGGGUUTRGRGAGGARGGGUUXGGRGUUURGUAAGRGAGGAGGARGARGGARGGARGGARGGARGGGURGRGGARGGGRGGARGGGAGGGAGRGAGRGGGRGRGGGGGRGGRGGURGGGAURGGTGGGGURGGGGRGGGGRGRGGRGAAURGGARGUUUU AAUUAUURGUUU179 TATTUAGAUTGGGAGUTGGGGTUAGAGTGTUTURGTUTURGGAGGGGGAUUTTUUAGAUUURGGGRGGGAGGGGUUUAUAGUAGGGTGUUURGGRGGUUUUAGGAGGGGGTURGGGUUTRGGGRGAAGGUTGRGGRGUUURGGAAGGAGUXGRGUARGUUUUTGUARGRGGTGUURGUTGRGGAGGUUARGGATURGGAGGTGAGAGRGUUUURGUUURGUTUUUTGUUUAGUURGRGRGRGGUURGGTURGGGGUTGGGGGRGGGRGUUAAGAGGGGRGURGGGRGU AGTGGGGRGGUU180 GGURGUUUUAUTGRGUURGGRGUUUUTUTTGGRGUURGUUUUUAGUUURGGAURGGGURGRGRGRGGGUTGGGUAGGGAGRGGGGRGGGGGRGUTUTUAUUTURGGATURGTGGUUTURGUAGRGGGUAURGRGTGUAGGGGRGTGRGXGGUTUUTTURGGGGRGURGUAGUUTTRGUURGAGGUURGGAUUUUUTUUTGGGGURGURGGGGUAUUUTGUTGTGGGUUUUTUURGUURGGGGTUTGGAAGGTUUUUUTURGGAGARGGAGAUAUTUTGAUUUUAGUTUUU AGTUTGAATA181 GRGAUTTRGTUTGGUUUUAAAAUUTTTGUUUTUUATTUUUUAGRGTUURGGAURGGTUUTTGUTUATUTUTUAGGGGUAAUAUUTGAUUUARGGGGUURGTUURGGAGUTUTUTTRGAUTURGGGAUUAGTTUUUAGUUUTTUAGTAUTXGGUATAURGGAAGGAGGATTUUTGUTUUUTTUTUTUUTUUTAGAUUURGAGGUTTGGAGUTTAUTUTUUAGATGAGAUTAAAAAGUUUTAAATTAATUUUUUTATAGRGUAUUUUTUUUTRGUAGRGUUUTGGTRGGGGGUU TUUATTGT 182AUAATGGAGGUUUURGAUUAGGGRGUTGRGAGGGAGGGGTGRGUTATAGGGGGATTAATTTAGGGUTTTTTAGTUTUATUTGGAGAGTAAGUTUUAAGUUTRGGGGTUTAGGAGGAGAGAAGGGAGUAGGAATUUTUUTTURGGTATGUXGAGTAUTGAAGGGUTGGGAAUTGGTUURGGAGTRGAAGAGAGUTURGGGARGGGUUURGTGGGTUAGGTGTTGUUUUTGAGAGATGAGUAAGGAURGGTURGGGARGUTGGGGAATGGAGGGUAAAGGTTTTGGGGUUAGA RGAAGTRGU 183UUARGGURGAGGGGURGGAGUTGUUTUTGTGRGAGGGAGUUUTGGURGRGGRGUAGGTAGGAURGGGGAAUUUAAGGAGGGRGGGAGGAAAGRGUUUUTGGTGGGGGAGGGGURGTGGRGGGGTUTGGGGGUUAAGGGUTURGGGURGAAXGAGGGGUAUARGURGGAGUAGUTUUAGUUTGGGAGUATTGGGRGTGAARGTGGGGGAAAGTAGGGAUUAGATTAUAGGATUAAGGUTTTUUURGGUTUAGAAUTGGATTGTTUAGTGUUUUTAGAAGGGGUAGRGGG TRGUUUARGRGA184 TRGRGTGGGRGAUURGUTGUUUUTTUTAGGGGUAUTGAAUAATUUAGTTUTGAGURGGGGAAAGUUTTGATUUTGTAATUTGGTUUUTAUTTTUUUUUARGTTUARGUUUAATGUTUUUAGGUTGGAGUTGUTURGGRGTGTGUUUUTXGTTRGGUURGGAGUUUTTGGUUUUUAGAUUURGUUARGGUUUUTUUUUUAUUAGGGGRGUTTTUUTUURGUUUTUUTTGGGTTUUURGGTUUTAUUTGRGURGRGGUUAGGGUTUUUTRGUAUAGAGGUAGUTURGGUUUUT RGGURGTGG 185GAGAARGGAGUTGUURGTGRGGGGTGRGRGUUAAGGRGGGGGAUAGGARGGGUURGGRGTUTGURGAGAUUTAGGUTGUURGUATTURGUTRGRGGUTUUUUTTUTGUUTUURGUTUTAGRGGRGURGGAGURGXGUTGGUUUURGUUURGUURGGUAGUTUUXGXGAGTUAGAGUURGGTGUUUUUAGAURGRGGGUAGTRGGGGUUTUATTURGGGUUAGAGUAGGAAAGAGUURGAUUUAUUTUUTRGGTTUTTUUAGGGAAUUUUTTUTRGGAGGGRGUUUTGG GUUTURGRGUUA186 TGGRGRGGAGGUUUAGGGRGUUUTURGAGAAGGGGTTUUUTGGAAGAAURGAGGAGGTGGGTRGGGUTUTTTUUTGUTUTGGUURGGAATGAGGUUURGAUTGUURGRGGTUTGGGGGUAURGGGUTUTGAUTXGXGGGAGUTGURGGGRGGGGRGGGGGUUAGXGRGGUTURGGRGURGUTAGAGRGGGAGGUAGAAGGGGAGURGRGAGRGGAATGRGGGUAGUUTAGGTUTRGGUAGARGURGGGUURGTUUTGTUUUURGUUTTGGRGRGUAUUURGUARGGGU AGUTURGTTUTU187 AGTAGGTGAAUTTGAGGTAGAAGRGGGARGTGTAUTTUTURGGUTGURGGTRGGGGTRGRGGUTUTRGTTGAGRGTGTUUURGAAGAUUTUUTTGGUUAGRGRGATGRGUURGUAUAUUATGATGRGRGUUAUARGURGGAATTTGGRGTXGGUUTGGTTGTRGRGUARGGTGGTGTAGGAGUUURGGTAGUUUAGRGTGAGGAAGUURGAGRGUTTGTUUTGRGRGURGURGURGURGUUAUUAURGTGRGUTUURGGGUURGAGGGUARGGRGGURGURGUUURGRGUA GUAGUAGRG 188RGUTGUTGUTGRGRGGGGRGGRGGURGURGTGUUUTRGGGUURGGGAGRGUARGGTGGTGGRGGRGGRGGRGGRGRGUAGGAUAAGRGUTRGGGUTTUUTUARGUTGGGUTAURGGGGUTUUTAUAUUAURGTGRGRGAUAAUUAGGUXGARGUUAAATTURGGRGTGTGGRGRGUATUATGGTGTGRGGGRGUATRGRGUTGGUUAAGGAGGTUTTRGGGGAUARGUTUAARGAGAGURGRGAUUURGAURGGUAGURGGAGAAGTAUARGTUURGUTTUTAUUTUAAG TTUAUUTAUT189 GURGRGAUUUUTGRGUTUUUAGUAGGGUUAAGAGGAUUTRGRGGGUAUUAGRGTURGGGRGGGAAGGGARGTGTGUUUAAGUUTRGUUTUUTGGUUUTUAGTGGGUTGGGARGUUUTTGATUAURGGRGUAGGAAAGAGGUTUUUUAGUUXGTGAGUTTRGTURGGGRGUUAGGGUAGGGATGGUTGGTGGTGTGUAUTGGAGAGUARGARGGTGARGUTGRGTGGGAAAGAGARGTGGGAAGGGUATAGURGGATTATUUAUTUAGUTUUAATTTTUTUUAAGRGUUA UTUAUUUUAUA190 TGTGGGGTGAGTGGRGUTTGGAGAAAATTGGAGUTGAGTGGATAATURGGUTATGUUUTTUUUARGTUTUTTTUUUARGUAGRGTUAURGTRGTGUTUTUUAGTGUAUAUUAUUAGUUATUUUTGUUUTGGRGUURGGARGAAGUTUAXGGGUTGGGGAGUUTUTTTUUTGRGURGGTGATUAAGGGRGTUUUAGUUUAUTGAGGGUUAGGAGGRGAGGUTTGGGUAUARGTUUUTTUURGUURGGARGUTGGTGUURGRGAGGTUUTUTTGGUUUTGUTGGGAGRGUAGG GGTRGRGGU 191CTGGGTTTGGAGTTGGGTAGRGRGGCCAGGTGARGGTCTCCTTCCCTGGGCRGTCAGGGCTGCAGGRGGCTGTGACACTCRGGAGGCCTRGACAGAGGGGTCCCAGGRGCAGRGTACCRGGGCTCCTCTTGCCTCAGRGGAGRGTCCCAXGGCAGTCCRGGACCRGRGCCTCTCCRGCCRGGCTCCTGCARGCCCAGRGAGCCTGCAGCCTGGGATCCTCRGCTCCTGCATCCRGGCTRGGGATGGGTCCRGGCTCCCCACCTCARGCRGGRGGGGCCACCCTTTCACTCRGCCTCCAT C 192GATGGAGGRGGAGTGAAAGGGTGGCCCRGCRGGRGTGAGGTGGGGAGCRGGGACCCATCCRGAGCRGGGATGCAGGAGRGGAGGATCCCAGGCTGCAGGCTRGCTGGGRGTGCAGGAGCRGGGRGGGAGAGGRGRGGGTCRGGGACTGCXGTGGGARGCTCRGCTGAGGCAAGAGGAGCCRGGGTARGCTGRGCCTGGGACCCCTCTGTRGAGGCCTCRGGAGTGTCACAGCRGCCTGCAGCCCTGARGGCCCAGGGAAGGAGACRGTCACCTGGCRGRGCTACCCAACTCC AAACCCAG 193UUUTUAUUTUTAGUUUUTRGGUTTUAGUTUAGGUUUUTRGGGGAGUATUUUTTGURGTGAGAUTGAUAGUUTTTGGGGGRGUAGGGTUUTGTTUTUTGRGUTUTAGUUUATUTGTGRGUAGAGUUTRGTTUUUAGGRGUUTGGAAUURGGXGGGUATTGARGTUAAGRGURGGRGGAGRGUTGUUTAUAGARGGTTGAUURGGGUUUTUUTUUAUAUUUUUTTUUTTUTTRGUUTUUTUUUTUTTTUUTGUARGGGGGUTRGGGUTUAUTATAAAAGGTGGGAGRGRGTGGT GUUUUAGU 194GUTGGGGUAUUARGRGUTUUUAUUTTTTATAGTGAGUURGAGUUUURGTGUAGGAAAGAGGGAGGAGGRGAAGAAGGAAGGGGGTGTGGAGGAGGGUURGGGTUAAURGTUTGTAGGUAGRGUTURGURGGRGUTTGARGTUAATGUUXGURGGGTTUUAGGRGUUTGGGAARGAGGUTUTGRGUAUAGATGGGUTAGAGRGUAGAGAAUAGGAUUUTGRGUUUUUAAAGGUTGTUAGTUTUARGGUAAGGGATGUTUUURGAGGGGUUTGAGUTGAAGURGAGGGGU TAGAGGTGAGGG195 TTGUTGUTAAATGTUAUAAAAGTUAUUTAAAGGUAUAGAGGAGGURGUTUTGTTTTTGRGAAAUTTGUTAAAATTAATUTGRGUTGGGUUAUTTGUAGAAAGUAGAAUUAUUTUURGUUUUUAUUTRGUUTUUAGURGURGGGGTTUAGGXGTTTGTGAAAGAUAGAAUUTTTGGGUTAGGGAUURGGGUAUTGGTGUTTRGAAGTURGAATURGURGGURGAGAAAARGAUAAGAGAAAGAAAATUUAGRGGGRGUTUTUTUUAGRGUUAGGURGGTGTAGGAGGGRGUT GGGGUTRGG 196URGAGUUUUAGRGUUUTUUTAUAURGGUUTGGRGUTGGAGAGAGRGUURGUTGGATTTTUTTTUTUTTGTRGTTTTUTRGGURGGRGGATTRGGAUTTRGAAGUAUUAGTGUURGGGTUUUTAGUUUAAAGGTTUTGTUTTTUAUAAAXGUUTGAAUUURGGRGGUTGGAGGRGAGGTGGGGGRGGGAGGTGGTTUTGUTTTUTGUAAGTGGUUUAGRGUAGATTAATTTTAGUAAGTTTRGUAAAAAUAGAGRGGUUTUUTUTGTGUUTTTAGGTGAUTTTTGTGAUATTTAG UAGUAA 197AURGAGRGUTGGAGGURGUTUUARGRGRGAGUTRGAAUUARGGAGGGUTUTUAGUTRGGAUAAGRGTRGUTGTUTAAGAGUTUUAAGUTUUARGGAAUTTTGATTTTATGUURGGGAGUURGGTUATUUAUTTUTGUATUTUAGAUAAGXGURGGAGGUTRGTGUAGUTTUUTRGRGTUUTUTTUUTGGARGRGGGGRGUUUAUUTTAGTUATAGUUUUTGGTUURGGUAUTGUTUARGURGTRGGTTTUUUUAUTGUUUAGGUTUUUTUAGUURGAAUUUTGRGTUTUTGT TUAUATTG 198UAATGTGAAUAGAGARGUAGGGTTRGGGUTGAGGGAGUUTGGGUAGTGGGGAAAURGARGGRGTGAGUAGTGURGGGAUUAGGGGUTATGAUTAAGGTGGGRGUUURGRGTUUAGGAAGAGGARGRGAGGAAGUTGUARGAGUUTURGGXGUTTGTUTGAGATGUAGAAGTGGATGAURGGGUTUURGGGUATAAAATUAAAGTTURGTGGAGUTTGGAGUTUTTAGAUAGRGARGUTTGTURGAGUTGAGAGUUUTURGTGGTTRGAGUTRGRGRGTGGAGRGGUUTUUA GRGUTRGGT199 AAUAGUAGGUTGAAUUAGAAUTAAGAGAAAATTGGGUAGAGAGAAGGUAATGGRGAGTUUAUUTAGGGGUTGGGGUTGRGGAGAGUTGUTGUTGUUUTTUATGUTUUTGGGGARGUTGTGRGAGUUAGGATURGGGUAGATURGUTAUTXGATGURGGAGGAGUTGGAUAAAGGUTUUTTRGTRGGUAAUATAGUUAAGGAUUTTGGGUTGGAGUUUUAGGAGUTGGRGGAGRGRGGAGTURGUATRGTUTUUAGAGGTAGGARGUAGUTTTTTGUUUTGAAUURGRGA AGRGGUAGUTT200 AAGUTGURGUTTRGRGGGTTUAGGGUAAAAAGUTGRGTUUTAUUTUTGGAGARGATGRGGAUTURGRGUTURGUUAGUTUUTGGGGUTUUAGUUUAAGGTUUTTGGUTATGTTGURGARGAAGGAGUUTTTGTUUAGUTUUTURGGUATXGAGTAGRGGATUTGUURGGATUUTGGUTRGUAUAGRGTUUUUAGGAGUATGAAGGGUAGUAGUAGUTUTURGUAGUUUUAGUUUUTAGGTGGAUTRGUUATTGUUTTUTUTUTGUUUAATTTTUTUTTAGTTUTGGTTUAG UUTGUTGTT201 GGTGTUURGRGAGUAGGTGTUUAGUAGURGRGRGUUUAGGRGUARGURGGGUAGUAGUTRGGGGTRGGRGTTGARGRGGTUUAGRGRGTAUAGUATGGUUTUUAGURGGTGUARGUUUTGUTUUTTUTTUAGUTGUURGUARGUURGGUUXGURGRGUUURGRGRGTGUAURGGGAAUAGGURGUUUAGRGTUAGGURGUURGUUAGGRGUAUAGAGUURGURGRGRGRGUUAGGUURGUUTGRGUUAGUUARGUUAGRGGUAGUAGRGUUARGAGUAGRGGUTUURG GGUTUTURGGGG202 UUURGGAGAGUURGGGAGURGUTGUTRGTGGRGUTGUTGURGUTGGRGTGGUTGGRGUAGGRGGGUUTGGRGRGRGRGGRGGGUTUTGTGRGUUTGGRGGGRGGUUTGARGUTGGGRGGUUTGTTUURGGTGUARGRGRGGGGRGRGGXGGGURGGGRGTGRGGGUAGUTGAAGAAGGAGUAGGGRGTGUAURGGUTGGAGGUUATGUTGTARGRGUTGGAURGRGTUAARGURGAUUURGAGUTGUTGUURGGRGTGRGUUTGGGRGRGRGGUTGUTGGAUAUUTGUTR GRGGGAUAUU203 GAAUUAGGGTAGGUATAGUAAAGGUAAUAGGTGGGARGGGGAGUUAGGGAGTGGAAARGURGGAGGTUUTUAGGUTGGGGATUURGGAGTUUTRGUTUAGATTURGATTURGGAGAAGGUUAATTURGGGAUUUTATUUATGTUUUATTUXGGGUAUUTTUUTUAAAAGTAGGGGTGGGAGGGGUATAAGURGGTUAAGTATTTUURGGGUUTTURGGAGUAATUTTGUUTAAGRGTTUATATTTAARGUATTGGAUTGGGAAAUATTTGGAGATUATTGATTAAGAAAT AUTUAAAUUT204 AGGTTTGAGTATTTUTTAATUAATGATUTUUAAATGTTTUUUAGTUUAATGRGTTAAATATGAARGUTTAGGUAAGATTGUTURGGAAGGUURGGGAAATAUTTGAURGGUTTATGUUUUTUUUAUUUUTAUTTTTGAGGAAGGTGUUXGGAATGGGAUATGGATAGGGTUURGGAATTGGUUTTUTURGGAATRGGAATUTGAGRGAGGAUTURGGGATUUUUAGUUTGAGGAUUTURGGRGTTTUUAUTUUUTGGUTUUURGTUUUAUUTGTTGUUTTTGUTATGUUTAUU UTGGTTU 205TTUTRGTRGGTAGRGUTUTGGUUTTGGGRGUURGURGUTTTGGUTATGGAGGTAUAGTGGURGRGTUURGGGGAGUUTGGGUUTUUTTGRGGUTURGAGUUTURGRGGRGUURGRGUUURGAUTUTURGGTUURGGUTUTUTUUAGGUUXGGUTGTUTURGGTUUUTUAUTGAUTTRGUUAGURGUAUUAUUUAUUTUTTTUTUURGUAGARGTAAAUURGAATARGUUURGGUUTTGGGUUTAGGGUUTRGGUUTRGUUTGGUTUTAUAGGATTRGGGGGRGGGGTAGGUR GGUAGAGU 206GUTUTGURGGUUTAUUURGUUUURGAATUUTGTAGAGUUAGGRGAGGURGAGGUUUTAGGUUUAAGGURGGGGRGTATTRGGGTTTARGTUTGRGGGAGAAAGAGGTGGGTGGTGRGGUTGGRGAAGTUAGTGAGGGAURGGAGAUAGUXGGGUUTGGAGAGAGURGGGAURGGAGAGTRGGGGRGRGGGRGURGRGGAGGUTRGGAGURGUAAGGAGGUUUAGGUTUUURGGGARGRGGUUAUTGTAUUTUUATAGUUAAAGRGGRGGGRGUUUAAGGUUAGAGRG UTAURGARGAGAA207 RGUAUAURGARGGUUTUURGGGTTRGGRGGGGARGAUURGAGUTAGGAGUURGRGGGGGURGTGGGAGUTGUTURGURGAGGTGGAUURGGGGRGUURGUAUUUUTUAUUTTUTTTURGTGGGGTURGGGGUUAGGGARGUAGRGGGGGAXGGRGGGUATUAGURGGGUAGRGUURGAGURGTUUUTGGURGGGTUUUUAUAUUTGRGTGUTRGGGUATUUUUAGGGTRGGGGGRGRGGTGAATRGTGGTUUURGUAGUURGGGGUURGUUAGGUURGRGGAGTUAGUU UAUUUTGGRGA208 TRGUUAGGGTGGGUTGAUTURGRGGGUUTGGRGGGUUURGGGUTGRGGGGAUUARGATTUAURGRGUUUURGAUUUTGGGGATGUURGAGUARGUAGGTGTGGGGAUURGGUUAGGGARGGUTRGGGRGUTGUURGGUTGATGUURGUXGTUUUURGUTGRGTUUUTGGUUURGGAUUUUARGGAAAGAAGGTGAGGGGTGRGGGRGUUURGGGTUUAUUTRGGRGGAGUAGUTUUUARGGUUUURGRGGGUTUUTAGUTRGGGTRGTUUURGURGAAUURGGGAGGU RGTRGGTGTGRG209 GAUUAAAAGGAGUTURGRGUUAGGURGGAGGUTGRGUURGTUAUTRGRGGTUURGGURGGGTUUUTGGRGRGTAGTUAGGURGUAURGUURGAGUUUUAURGRGRGUUUAUUURGGURGGGTGRGTURGGUTRGRGGURGTUUUTUURGXGAUUTGTGGUURGGGGUTGUTGRGGGRGUURGGGGAAGAGAGGRGGGGGURGRGGGGGGUAGGAGGAGRGGUTGRGGURGGUAUAGRGUUAGGGRGAGTGAGGRGGGTGGRGRGGGGGAGGRGGRGGAGTAAAGAGAG GURGURGGUTGG210 UUAGURGGRGGUUTUTUTTTAUTURGURGUUTUUUURGRGUUAUURGUUTUAUTRGUUUTGGRGUTGTGURGGURGUAGURGUTUUTUUTGUUUUURGRGGUUUURGUUTUTUTTUUURGGGRGUURGUAGUAGUUURGGGUUAUAGGTXGRGGGAGGGARGGURGRGAGURGGARGUAUURGGURGGGGTGGGRGRGRGGTGGGGUTRGGGRGGTGRGGUUTGAUTARGRGUUAGGGAUURGGURGGGAURGRGAGTGARGGGRGUAGUUTURGGUUTGGRGRGGAG UTUUTTTTGGTU211 GURGGUAUUTGURGUTURGUURGGGATTAGGGAGUTUURGGUTTGTGGGGGGTGRGGGRGGURGGTGGTUUAGUTRGUURGUUURGGURGAGARGUTGGGUURGGUUUUUAUAGGUTGRGUUTGARGGGAURGGRGGGAGGUUTTTGTTXGUAGUURGGAGGUUUUAGGUTUUAAAUUAUAUTRGGGUTGRGGGGAGRGAGRGRGGGRGTTGURGUTAATTGUTGUTAATTTTGTTUUATTAAUTTTATTTAUTUTTGAAUUUUTTAATTGTTTTUUAUTTATTTTTAGTAAT TTTATAA212 TTATAAAATTAUTAAAAATAAGTGGAAAAUAATTAAGGGGTTUAAGAGTAAATAAAGTTAATGGAAUAAAATTAGUAGUAATTAGRGGUAARGUURGRGUTRGUTUUURGUAGUURGAGTGTGGTTTGGAGUUTGGGGUUTURGGGUTGXGAAUAAAGGUUTUURGURGGTUURGTUAGGRGUAGUUTGTGGGGGURGGGUUUAGRGTUTRGGURGGGGRGGGRGAGUTGGAUUAURGGURGUURGUAUUUUUUAUAAGURGGGAGUTUUUTAATUURGGGRGGAGRGGU AGGTGURGGU 213TUAGUAURGGGGAUAGUTUUTGUUTRGUUAAUTTRGGUAGUTUTTUTRGATGTUTRGURGRGGGUTGTGUUTUAUAGGUTARGGGAGGGGAGAGTGTUTUUUUARGURGGGGTUURGGUUTUTGGGTTTTAGGGAGRGRGAATGGGTUTURGAUAGUAARGGGAGUAGURGGTGGRGUUTUAGGUTGRGGTGGUAARGAGUURGAUTGUAUTARGGUTTTGRGGGAURGRGUUAGRGRGGAGGAGAURGAGUUUATUTUAGRGGGURGGGURGGAUUUAGGTGAGRGGUU UTGGUTUUUU 214GGGGAGUUAGGGURGUTUAUUTGGGTURGGUURGGUURGUTGAGATGGGUTRGGTUTUUTURGRGUTGGRGRGGTUURGUAAAGURGTAGTGUAGTRGGGUTRGTTGUUAURGUAGUUTGAGGRGUUAURGGUTGUTUURGTTGUTGTRGGAGAUUUATTRGRGUTUUUTAAAAUUUAGAGGURGGGAUUURGGRGTGGGGAGAUAUTUTUUUUTUURGTAGUUTGTGAGGUAUAGUURGRGGRGAGAUATRGAGAAGAGUTGURGAAGTTGGRGAGGUAGGAGUTGTUU URGGTGUTGA215 TTUUTURGAGGUURGUAGGGAGGGGGRGGTGRGGAATGGATGRGURGAGRGGGUAGRGUTUAGUUTUTRGUTUAUAUUUUUAGUAGGUAGURGRGTUUURGTGURGGUATUUTRGUTGURGURGGUTUUUTRGGRGUUUURGGGUXGUTUUUUAXGRGRGRGURGGGAUUTGUARGAGUUUUUTRGTRGAUTRGGAGRGRGATUTGGGRGRGTGUUTUUUTGTUUTTGTUUTUTGUTUTRGTUTGGGGARGTGTGUUURGUAUUUUUTGUAURGRGRGUTUUTUTAUUUUTU URGUTUUU 216GGGAGRGGGAGGGGTAGAGGAGRGRGRGGTGUAGGGGGTGRGGGGUAUARGTUUUUAGARGAGAGUAGAGGAUAAGGAUAGGGAGGUARGRGUUUAGATRGRGUTURGAGTRGARGAGGGGGUTRGTGUAGGTUURGGRGRGRGXGTGGGGAGXGGUURGGGGGRGURGAGGGAGURGGRGGUAGRGAGGATGURGGUARGGGGARGRGGUTGUUTGUTGGGGGTGTGAGRGAGAGGUTGAGRGUTGUURGUTRGGRGUATUUATTURGUAURGUUUUUTUUUTGRGG GUUTRGGAGGAA217 AAGGGGAGGGRGGGGURGAUURGGAUUUTUUAGGGAARGUUUTTGAGTAAUTRGRGUAUTTGGGAUUUATTUUUAUUTAGGAUUATAGGUTUAAGATGGUUTGGTGGATGURGURGGUARGRGUUTUTUUTUTGGRGGGAAUXGAAGGRGUXGGTAGGTTTTUAUAUTTGUAGURGATRGGUTAAGAGAARGRGGGATTUAGURGAGAAGUUAUTGGGAGUURGAGGAGRGGAGUAGAGGUAUUUAGGUAGUUTGRGRGGAGAAATRGGATRGGUTAGGARGGUUTGUA GUUUUTGRGRG218 RGRGUAGGGGUTGUAGGURGTUUTAGURGATURGATTTUTURGRGUAGGUTGUUTGGGTGUUTUTGUTURGUTUUTRGGGUTUUUAGTGGUTTUTRGGUTGAATUURGRGTTUTUTTAGURGATRGGUTGUAAGTGTGAAAAUUTAUXGGRGUUTTXGGTTUURGUUAGAGGAGAGGRGRGTGURGGRGGUATUUAUUAGGUUATUTTGAGUUTATGGTUUTAGGTGGGAATGGGTUUUAAGTGRGRGAGTTAUTUAAGGGRGTTUUUTGGAGGGTURGGGTRGGUUURGUU UTUUUUTT 219TGTGGRGGGGGUTTGGAGUTGUTGAGAGURGAGAGGRGUAGAGRGUAAGUTGGUAGGUTGGGUTGUTATUURGGRGRGUAGATGUUURGURGUUAGTRGAGRGRGAAUATUTUTURGGAAUATRGATUTATUAUUTUUUTTTAAGGAUUXGGAURGGGAAATTTUUATTTTUTGTTTTGGGAATAAGAAATAAAAGRGAUUAAGUTUTTGUUUTAATTTUUUUURGRGGGUUUTTUUARGRGGGUTGGRGGGATUAGAAGGARGGGTURGAGUTRGGGGGRGRGGGGTTUU TGTGAAUTU 220GAGTTUAUAGGAAUUURGRGUUUURGAGUTRGGAUURGTUUTTUTGATUURGUUAGUURGRGTGGAAGGGUURGRGGGGGGAAATTAGGGUAAGAGUTTGGTRGUTTTTATTTUTTATTUUUAAAAUAGAAAATGGAAATTTUURGGTUXGGGTUUTTAAAGGGAGGTGATAGATRGATGTTURGGAGAGATGTTRGRGUTRGAUTGGRGGRGGGGUATUTGRGRGURGGGATAGUAGUUUAGUUTGUUAGUTTGRGUTUTGRGUUTUTRGGUTUTUAGUAGUTUUAAGUUU URGUUAUA 221UAGUUUTTGGUTTTUURGUTTUAGGUAAAATUTTUUUTUUTTUUTUTTTTTTUTGGGGUUTTUUUUARGAUUUUTUTTUUTAGUUUTTUTGATURGTUUUUTGATGUATGAUTGGGGUUAURGGAGGGGUTGAUUUTUURGGAGAGUUUXGTRGGTUUTGGTGGTUTTGGGAURGGAGAGRGAUAGATGTGGAAAURGAGGUUUUTUAGTGAAGAGUTGUUAGGGTGGTRGUUTTAGAGUAAAGGRGTTUUTUATTURGUUTGRGUAGUTGUUUUTURGUURGGUTGURGUUU UAGUURG 222RGGGUTGGGGRGGUAGURGGGRGGAGGGGUAGUTGRGUAGGRGGAATGAGGAARGUUTTTGUTUTAAGGRGAUUAUUUTGGUAGUTUTTUAUTGAGGGGUUTRGGTTTUUAUATUTGTRGUTUTURGGTUUUAAGAUUAUUAGGAURGAXGGGGUTUTURGGGAGGGTUAGUUUUTURGGTGGUUUUAGTUATGUATUAGGGGARGGATUAGAAGGGUTAGGAAGAGGGGTRGTGGGGAAGGUUUUAGAAAAAAGAGGAAGGAGGGAAGATTTTGUUTGAAGRGGGAAA GUUAAGGGUTG223 GGGAAAGGGGGGAGAGGGAGAGGAGGRGRGGGGTGGGGGAGGGGAGTGAGUAGGGAGURGGGAGAGGGAGGAGGGGRGGGAAUUAGGGGAGRGGUURGAAUUURGTTTGGTURGGAUUURGUAGUUAURGUTGGGTUTRGURGURGGGTXGUUUTTRGRGTGGAGATURGGTURGRGUUUUUTUUURGGTUTUUTUUUUTUUUUTUUUUTURGUUUUUUTUUUTURGGUUUAUAUAGUUTUTTUUAGAAAGAAGTUAUTUTAGAGURGRGRGAUUUAGUUUUAGAGTU RGURGGGGTURG224 RGGAUUURGGRGGAUTUTGGGGUTGGGTRGRGRGGUTUTAGAGTGAUTTUTTTUTGGAAGAGGUTGTGTGGGURGGAGGGAGGGGGGRGGAGGGGAGGGGAGGGGAGGAGAURGGGGAGGGGGRGRGGAURGGATUTUUARGRGAAGGGXGAUURGGRGGRGAGAUUUAGRGGTGGUTGRGGGGTURGGAUUAAARGGGGTTRGGGURGUTUUUUTGGTTUURGUUUUTUUTUUUTUTUURGGUTUUUTGUTUAUTUUUUTUUUUUAUUURGRGUUTUUTUTUUUTUTU UUUUUTTTUUU225 RGGTURGRGUUUUUTUUURGGTUTUUTUUUUTUUUUTUUUUTURGUUUUUUTUUUTURGGUUUAUAUAGUUTUTTUUAGAAAGAAGTUAUTUTAGAGURGRGRGAUUUAGUUUUAGAGTURGURGGGGTURGUUUAURGGGTUTUUTGXGXGUUUUTUUURGUUUUTUUURGGGUAUAGUURGTTUARGAAAUUTAAGGRGURGGUUARGRGUUAUUTUUUURGGGURGGGGTUTUUTRGGTUUURGRGRGGGRGUTGGTTUTUURGGGTGGGRGGUAGUUURGUUUT GTGUUUTUUTGG226 UUAGGAGGGUAUAGGGRGGGGUTGURGUUUAUURGGGAGAAUUAGRGUURGRGRGGGGAURGAGGAGAUUURGGUURGGGGGAGGTGGRGRGTGGURGGRGUUTTAGGTTTRGTGAARGGGUTGTGUURGGGGAGGGGRGGGGAGGGGXGXGUAGGAGAUURGGTGGGRGGAUUURGGRGGAUTUTGGGGUTGGGTRGRGRGGUTUTAGAGTGAUTTUTTTUTGGAAGAGGUTGTGTGGGURGGAGGGAGGGGGGRGGAGGGGAGGGGAGGGGAGGAGAURGGGGAGG GGGRGRGGAURG227 GRGGTRGGRGUUUUARGUTUUUTGAARGUUUUUUAGGRGGUAUUAGTGURGGGUAGAGTUUUUTRGGRGGURGRGGGRGTUAAATRGAUAUUTGAUTUUAUAGUTUUUTTUUUTUTUUTUTTUTTUUAUUTUUURGGGGGTUUAGGUAUXGURGTGRGTUUAUTUURGGTUTUUARGGUTTAGGUAGARGGAGTGGGGGAUTURGGGGAUURGRGRGTUUTUTUUTUUTRGGUUUTGGRGGGAGGAGURGGGUTGGGGTTTURGRGGGURGRGGRGRGTTTTAGGGUTGU RGGGGAUTGU228 GUAGTUUURGGUAGUUUTAAAARGRGURGRGGUURGRGGAAAUUUUAGUURGGUTUUTUURGUUAGGGURGAGGAGGAGAGGARGRGRGGGTUUURGGAGTUUUUUAUTURGTUTGUUTAAGURGTGGAGAURGGGAGTGGARGUARGGXGGTGUUTGGAUUUURGGGGAGGTGGAAGAAGAGGAGAGGGAAGGGAGUTGTGGAGTUAGGTGTRGATTTGARGUURGRGGURGURGAGGGGAUTUTGUURGGUAUTGGTGURGUUTGGGGGGRGTTUAGGGAGRGTGGGG RGURGAURGU229 GUUUTUUTURGTGUTGGGUUTGTUUTAUUTUUAGGGRGGAGGRGRGGGUTUTGRGTURGGAGGRGUUTRGGGRGGUAGUTURGGTGGGGURGRGTUTGGTGRGGGGUURGGGAUUUAGUAGGGUAGUUXGGGATGGAGUUAGGRGGGAGUXGARGGAGURGUTTAUAUUURGUXGURGGTGTRGURGRGUTTUTUUTTUURGGGGAUUAURGGGTUUUTGGRGGURGURGURGURGUTGURGRGGUURGGGAAGUTGRGGUUTAUAGUAGTGGRGGRGGAGRGGRGGGT GRGGGUUTGGU230 GUUAGGUURGUAUURGURGUTURGURGUUAUTGUTGTAGGURGUAGUTTUURGGGURGRGGUAGRGGRGGRGGRGGURGUUAGGGAUURGGTGGTUUURGGGAAGGAGAAGRGRGGRGAUAURGGXGGXGGGGTGTAAGRGGUTURGTXGGUTUURGUUTGGUTUUATUUXGGGUTGUUUTGUTGGGTUURGGGUUURGUAUUAGARGRGGUUUUAURGGAGUTGURGUURGAGGRGUUTURGGARGUAGAGUURGRGUUTURGUUUTGGAGGTAGGAUAGGUUUAGU ARGGAGGAGGGU231 AGAAAAGUAGRGARGTGGRGTTUAUUURGUTGUAGAAUTRGGAUUAUTRGGGUTRGGTGUAGGGATTGGUTUUAGGUTTGURGTRGGGGTRGGGAGURGAGGARGAGGAGGRGGURGGGGGRGGUTGUTGUURGGARGGRGGRGGUTGUTXGRGUTGUTGUTGUTGUTGRGURGGGAGTGGRGGUTURGRGGGUTRGGGRGGUTURGGRGGRGTRGURGGUURGGGRGGRGGRGGGGRGGGUTRGGUTGRGUTGTGUUTGRGUUTGGGUAGGGAGUAGRGGRGUTAUTUA UTGTGGGAUT232 AGTUUUAUAGTGAGTAGRGURGUTGUTUUUTGUUUAGGRGUAGGUAUAGRGUAGURGAGUURGUUURGURGURGUURGGGURGGRGARGURGURGGAGURGUURGAGUURGRGGAGURGUUAUTUURGGRGUAGUAGUAGUAGUAGRGXGAGUAGURGURGURGTURGGGUAGUAGURGUUUURGGURGUUTUUTRGTUUTRGGUTUURGAUUURGARGGUAAGUUTGGAGUUAATUUUTGUAURGAGUURGAGTGGTURGAGTTUTGUAGRGGGGTGAARGUUARGT RGUTGUTTTTUT233 GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAATGUATTTAGAGGARGGGAGUTGUUTUTAGGGAGAAAGGGGAGGGGGGAGGGGAGAAGAAAAAGGAAAGAGRGRGTTTUUTRGURGGGGTTAURGGGGTGGXGRGGGUTTRGRGGURGAGGGGUTGURGGUURGGUURGUAGUTUUTRGRGGURGGUTGGGAAGTGGRGGGTURGGUTAGTURGRGGGRGGGRGGAGRGRGURGTRGUTGUUAGTGTUUUUUTTTAAUATTUUTTUUURGAGUUT TTUUUT 234AGGGAAAGGUTRGGGGAAGGAATGTTAAAGGGGGAUAUTGGUAGRGARGGRGRGUTURGUURGUURGRGGAUTAGURGGAUURGUUAUTTUUUAGURGGURGRGAGGAGUTGRGGGURGGGURGGUAGUUUUTRGGURGRGAAGUURGXGUUAUUURGGTAAUUURGGRGAGGAAARGRGUTUTTTUUTTTTTUTTUTUUUUTUUUUUUTUUUUTTTUTUUUTAGAGGUAGUTUURGTUUTUTAAATGUATTAUAUAUAUAUAUAUAUAUAUAUAUAUAUAUAUAUAUA UAUAUAUAUAU235 UTUUTGGGRGAUUTAAAUTTRGATAAUTGUTGGAAAGGTUUAGUAGGAGAGGAGGGRGAGGAGGGGRGGAGUUAGGAGGUURGGUUURGUUUAURGRGRGUURGUUTUTUTUTGUAGAUUAURGGUTAGAGUAGGAGUARGAGUTUTTUXGUTGTTTUUAGGATUUUTGURGGUTGGGUARGRGUUAAAAGUAGUUUTGGGUUUTGGGTATRGRGUTTGGGGGGAGGGTAUUUURGURGGUTGGGUARGRGUUAAGAGUAGUUUTGGGUUUTGGGTATRGTGUTTAGGG GGAGGGTATRG236 RGATAUUUTUUUUUTAAGUARGATAUUUAGGGUUUAGGGUTGUTUTTGGRGRGTGUUUAGURGGRGGGGGTAUUUTUUUUUUAAGRGRGATAUUUAGGGUUUAGGGUTGUTTTTGGRGRGTGUUUAGURGGUAGGGATUUTGGAAAUAGXGGAAGAGUTRGTGUTUUTGUTUTAGURGGTGGTUTGUAGAGAGAGGRGGGRGRGRGGTGGGRGGGGURGGGUUTUUTGGUTURGUUUUTUUTRGUUUTUUTUTUUTGUTGGAUUTTTUUAGUAGTTATRGAAGTTTAGGTR GUUUAGGAG237 GGRGURGGGRGUTGGGUTTAUAGUAGAGURGRGGGURGRGGGGTRGGAAAGTUUTTURGGGGRGGGGURGUAGRGGUUTUTTUURGUAGUUUUTRGGGUURGGGUUURGGTGGAARGGAAAUUTUUUUUTAUUURGGGAGGGGUTGUUAGXGGGUTGGGGGTGRGAAAARGGRGGUAGGAGRGGGRGAGGGGUURGGGURGRGUAUTTTGRGUUTGGGTTTGRGRGURGRGGURGRGGGAGTUURGRGRGGAURGGURGGARGUURGGUUTUUUUUAGUUUUAGUTTTT TGTGTGTGTGT238 AUAUAUAUAUAAAAAGUTGGGGUTGGGGGAGGURGGGRGTURGGURGGTURGRGRGGGAUTUURGRGGURGRGGRGRGUAAAUUUAGGRGUAAAGTGRGRGGUURGGGUUUUTRGUURGUTUUTGURGURGTTTTRGUAUUUUUAGUUXGUTGGUAGUUUUTUURGGGGTAGGGGGAGGTTTURGTTUUAURGGGGUURGGGUURGAGGGGUTGRGGGAAGAGGURGUTGRGGUUURGUUURGGAAGGAUTTTURGAUUURGRGGUURGRGGUTUTGUTGTAAGUUUAG RGUURGGRGUU239 AGGUAUAGAAAGGRGUAGURGUTAGUUAGAGUURGUAUAGAGUUAGAGUUAGGTUUUUAUTGGRGUAGATGGGGGAUTGUAGGUAUAGUAGTAGUUARGRGGGTGTAAARGTAGAGURGRGTGAAUURGGGTTGTGGGATUUUAGGUUUXGAUUAGUUUUUATUUURGGUTGGUATUTRGGTUURGGGGAGTUTUAGUTTUUUTTTUTGUAGAATGGGUTGGAGGRGUUUUUUAUAGGUURGUUUAGGRGUUUUURGGGGUUAGRGUUUUTUUUUUAUUTGUUURGUU UUUAUURGRGGG240 UURGRGGGTGGGGGRGGGGUAGGTGGGGGAGGGGRGUTGGUUURGGGGGGRGUUTGGGRGGGUUTGTGGGGGGRGUUTUUAGUUUATTUTGUAGAAAGGGAAGUTGAGAUTUUURGGGAURGAGATGUUAGURGGGGATGGGGGUTGGTXGGGGUUTGGGATUUUAUAAUURGGGTTUARGRGGUTUTARGTTTAUAUURGRGTGGUTAUTGUTGTGUUTGUAGTUUUUUATUTGRGUUAGTGGGGAUUTGGUTUTGGUTUTGTGRGGGUTUTGGUTAGRGGUTGRGUUT TTUTGTGUUT241 GGGGURGRGGAGTRGGGTGAGGRGGRGGRGGUTGRGGRGGTGGGGURGGGRGAGGTURGUTGRGGTUURGGRGGUTURGTGGUTGUTURGUTUTGAGRGUUTGGRGRGUUURGRGUUUTUUUTGURGGGGURGUTGGGURGGGGATGUAXGRGGGGUURGGGAGUUATGGTURGUTTRGGGGARGAGUTGGGRGGURGUTATGGGGGUUURGGRGGRGGAGAGRGGGUURGGGGRGGRGGGGURGGRGGGGRGGGGGGUURGGGTUURGGGGGGUTGUAGUURGGUUAG RGGGTUUTUTA242 TAGAGGAUURGUTGGURGGGUTGUAGUUUUURGGGAUURGGGUUUUURGUUURGURGGUUURGURGUUURGGGUURGUTUTURGURGURGGGGUUUUUATAGRGGURGUUUAGUTRGTUUURGAAGRGGAUUATGGUTUURGGGUUURGXGTGUATUUURGGUUUAGRGGUUURGGUAGGGAGGGRGRGGGGRGRGUUAGGRGUTUAGAGRGGAGUAGUUARGGAGURGURGGGAURGUAGRGGAUUTRGUURGGUUUUAURGURGUAGURGURGURGUUTUAUURG AUTURGRGGUUUU

Compositions for Detecting Methylation

Also provided herein are probes and primers that are complementary toone or more of SEQ ID NOS: 1-242. In embodiments, pairs of primerscomplementary to nucleotide sequences on either side of a methylationsite of interest listed in Table 1 are provided. In embodiments, aplurality of probes and/or primers are provided to detect and/or amplifya polynucleotide (e.g., a polynucleotide obtained by bisulfate treatmentof DNA) comprising a methylation site of interest. In embodiments, aprobe or primer is complementary to a polynucleotide sequence thatencompasses the methylation site of interest. In embodiments, the probeor primer is complementary to a sequence that is proximal to themethylation site of interest (e.g., within 1000, 900, 800, 700, 600,500, 400, 300, 200, 100, 75, 50, or 25 nucleotides of the methylationsite of interest in a genomic or bisulfate-treatment-derivedpolynucleotide).

In embodiments, a deoxyribonucleic acid selected from SEQ ID NO:243 toSEQ ID NO:356 is included. In embodiments, the deoxyribonucleic acidselected from SEQ ID NO:243 to SEQ ID NO:356 is hybridized to acomplementary DNA sequence having uridine or cytosine. In embodiments,each of the nucleic acids is different. In embodiments, each of thenucleic acids does not simultaneously have the same sequence selectedfrom SEQ ID NO:243 to SEQ ID NO:356. SEQ ID NOS: 243 to 356 are listedin Table 5 below.

TABLE 5 SEQ ID NOS: 243 to 356 SEQ ID NO: Sequence 243GTTTTTTTTTTAAAGTAGTTTTT 244 CTCCTACAACCCCCTTCC 245TTTTGAGTAGTTGGAGTTATAG 246 TAAATTCATCCTCTACCTATTA 247ATGGTATGAATTAATTAATTTGA 248 CTCCCTAAAAAAAAAAATAAA 249TATTTGTTTGATTTTAATTATATTT 250 AAACTCTACTCTCTAAACCTTTC 251ATTTTATTGATTATGTTTAGTTGATTA 252 AAAAATACCCCAAAAACAA 253GTGGAAGGGAAAAAAAAAGAG 254 ACAAACTCCCCTATACCTCAAATA 255TTTTTTAAATGGTGAAATAT 256 AATTTTACTTCTTCTTCTTATC 257TGGTAATAATTGGAGGAATTG 258 ACAAATAAAATCATAAAAAATAACAAAC 259GATTTTTTGATTTGAAAATAGTT 260 AATCTCAACCCCAAACTC 261TTAGGTAAAGATTTGGTTTTAGAA 262 AACTTATTATATAAATTATAAAAAAATAAA 263GGAGGTGTTTTTTAGTAAGTTTG 264 CCTCATACCTATAACCTACACTCA 265AAGGGTTGTTATGTTAGTGTAGT 266 CCCAAAAATAAATAATTTAACTA 267ATTTTTGTTTTAATATGGAGTTG 268 AAACCTAAATCTACACTTAAACATC 269AGGGTTAGGGTTTTTTTG 270 AAATCTCTTTATTACTCATTTTCTATA 271GTTATTTATATTTTTGAGTATTAAGAGTT 272 ACCAACAAATACAACACCTTCT 273GGTTTTTTAGTTTTATGAATTATTTA 274 AACCCCTCTACAACCTACTAC 275GATGAGGAAGTTGAGGTATAG 276 CTCCAACCCATTCTACAA 277 GAGGGGATTGAGTAGGTGAATAG278 CAAAACAAAAAAAAAATAAAAAAAAAACT 279 AAGGAGAGGAGAAGAGGTATAG 280AATAATAAAAAAAAACTAAATTCAAAC 281 TTGGGGTTTAGGGATTTAG 282AATAAAATATACCTCCTTTCAAACTAA 283 AAAGGTTAAATTAAAAATTTTTTTAT 284CTCCCAACTATACTTCTTAATCTC 285 GGTTTAGAGTTATTGAATAAATGAAGTG 286CAAAATCAAATTCTCCAACAAA 287 TGATGGAGGAAGTTTTTG 288 AACCCTAAACTAAACAACCC289 TTAAGGATTTAGATATTTTGTAAT 290 AATAAATTTATAAATTTACTCTCTTAC 291AATGTTTTGGAGATTAGTAATAT 292 TCTTCCTAAAAAAAAAATAAA 293GGGAATAATGAGGAGGAGA 294 AAAAATACAATAAAAAATCTTAAAATAAA 295GGTAGGGGATTGGGATAG 296 AACAAAACAAAACTATAAAATTAAACTAA 297GGTTAGGAGATTAGGGATTG 298 AAAAAAAAAAACAACTTAAAAAAC 299TGTTTATTTTAGAGGTGTTTATTT 300 ACCCTAATCCAATATCCC 301 GGTTGGTTAGGTTGTTATTT302 CAAACACCACATACTTATTC 303 TTTGGGTTTAGAAAGTTG 304CAAAAACTAAAAAAAAAATAAC 305 TTTTGAGTTTGGTTTAGTT 306 CCTAAAAAAAATAAAAATCC307 GGAATTTGGAGGGTAAAA 308 CTAATCTCTATCCTATATATTTCTTTATATT 309TATTATATTGTTATGTTGATT 310 ATATTAATTTCTTCCAACTAA 311TGGGATTGGTAAGTAGGTATT 312 AAAAAAAAAAACCTATAACCTATAAA 313ATGGTTTTTGTTTGTAGAC 314 ATTATTATTATTTATTTATTTATTATCA 315TTTTTTTGAAAGTTAGTGAATTTATTTATT 316 ACCCATCTCCCCACACAC 317TTGGGGTTTAAAGGTATTAG 318 AAAACAAAAACTACTAAAAAAAAAT 319TTGGTTAAGGAAGAAAGGAGTAG 320 CACCCCCTTCAAAAAAAA 321 TGGGGTTTTTTGGTTTTTT322 CCACCTCACAAACACACAC 323 AGAAAGGTATTTGTTTTTAGTAAA 324TTACAAAATAAAACCAACCTAT 325 GAGTTTTTTTTTTTATTTAGTTTT 326CCTACCCCCTAATATCTACA 327 TTTTTTTAATATTTGTGAATTAT 328AAACAACAACCCTAACTATC 329 ATGGAAGTTGGAGTTGAGAAG 330ACACCTAAAACAAAAATAAAAAAATC 331 ATTAGTTTTTAGTTTTTTAGTATT 332TCCCCTTAACATTAAATC 333 TTGAGATTAGATTAGAGTTTATTT 334AAAAAAACCTTAATCCTATAAT 335 TTTTTAGTAGGGTTAAGAGGATTT 336TCCAATACACACCACCAA 337 TTTGAATGGTAGAGGAAATAGTT 338ACCCAAAAATTCTATCTTTCAC 339 TTTATGGGTTTTTATTTTAGTA 340AAAAATATTCCAATATAAACAAA 341 AGAGTTGTTGTTGTTTTTTATGT 342CAACCCAAAATCCTTAACTATA 343 AAAAGAATTAGGGTAGGTATAGT 344CTTAATCAATAATCTCCAAATAT 345 TTTGGTTATGGAGGTATAGT 346AAAAAAAAAATAAATAATAC 347 ATTTTATTTGGGGATTTTTAATA 348TAAAACAAAATTAACAACAATTAAC 349 TAAGATGGTTTGGTGGATGT 350AAATCTCTCACTCACCCTTTC 351 GGTTTGGAGTTGTTGAGAG 352AAAAAAATTAAAACAAAAACTTAATC 353 ATTTTTTTTTTTAGTTTTTTTGAT 354CACCCTAACAACTCTTCACT 355 GGGGAGAAGAAAAAGGAAAG 356AAAAAAAATATTAAAAAAAAACACTAACA

In embodiments, aspects include a deoxyribonucleic acid selected fromSEQ ID NO:243 to SEQ ID NO:356, hybridized to corresponding acomplementary DNA sequence having uridine or cytosine, and in a complexwith an enzyme, e.g., a thermostable DNA polymerase. In embodiments, thethermostable DNA polymerase is Taq DNA polymerase.

In some aspects, the method includes deoxyribonucleic acid that has asequence that is at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%,75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical or homologous to a nucleic acid having a sequence of atleast one of SEQ ID NO:243 to SEQ ID NO:356.

Kit for Detecting Methylation Level of a DCIS Cell Mass

Also provided is a kit including a plurality (e.g., at least about 10,20, 40, 50, 100, 110 or 118) nucleic acids each independently comprisingone sequence selected from SEQ ID NO:243 to SEQ ID NO:356, in which thenucleic acids do not simultaneously include the same sequence.

In some aspects, the kit includes deoxyribonucleic acid that has asequence that is at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%,75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical or homologous to a nucleic acid having a sequence of atleast one of SEQ ID NO:243 to SEQ ID NO:356.

The kit provided herein may include enzymes, reagents for deamination ofcytosine, buffers, vials, control DNA, devices for collecting DCIStissue samples, reagents for isolating DNA, reagents for labeling DNA,labels, or any combinations thereof.

The kit provided herein may include enzymes such as thermostable DNApolymerase enzymes, restriction enzymes, and combination thereof.

In embodiments, the kit(s) may further include enzymes, reagents fordeamination of cytosine, buffers, vials, control DNA, devices for tissuesamples, or reagents for labeling DNA, or any combinations thereof.

In embodiments, a kit provided herein may include a solid carriercapable of adsorbing the nucleic acids containing in a sample of a bodyfluid, for example blood (whole blood, plasma, or serum). The kit mayalso contain other components for example, reagents, in concentrated orfinal dilution form, chromatographic materials for the separation of thenucleic acids, aqueous solutions (buffers, optionally also inconcentrated form for final adjusting by the user) or chromatographicmaterials for desalting nucleic acids which have been eluted with sodiumchloride.

In embodiments, a kit provided herein includes materials for purifyingnucleic acids, for example, inorganic and/or organic carriers andoptionally solutions, excipients and/or accessories. Such agents areknown and are commercially available. For solid phase nucleic acidisolation methods, many solid supports have been used including membranefilters, magnetic beads, metal oxides, and latex particles.

In addition, a kit can also contain excipients such as, for example, aprotease such as proteinase K, or enzymes and other agents formanipulating nucleic acids, e.g., at least one amplification primer,nucleic acid bases (A, T, G, C, and/or U), and enzymes suitable foramplifying nucleic acids, e.g., DNase, a nucleic acid polymerase and/orat least one restriction endonuclease. Alternatively, a commercialpolymerase chain reaction kit may be used to amplify the DNA samples.

Exemplary Techniques for Detecting Specific Sequences

Specific sequences, such as the sequences listed in Table 1 (or portionsthereof containing a methylation site of interest), can be detected bynumerous methods that are well-established in the art (e.g., PCR-basedsequence specific amplification, isozyme markers, northern analysis,sequence specific hybridization, and array based hybridization). Inembodiments, the presence or absence of methylation is determinedthrough nucleotide sequencing of the site of interest (e.g., the site inbisulfite-treated DNA or an amplicon thereof). Any of these methods arereadily adapted to high throughput analysis.

Some techniques for detecting specific sequences utilize hybridizationof a probe nucleic acid to nucleic acids corresponding to themethylation site of interest (e.g., amplified nucleic acids producedusing bisulfite-treated DNA as a template or the bisulfite-treated DNAitself). Hybridization formats, including, but not limited to: solutionphase, solid phase, mixed phase, or in situ hybridization assays areuseful for sequence detection. A non-limiting guide to the hybridizationof nucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes Elsevier, N.Y., as well as in Sambrook, Berger and Ausubel.

Nucleic acid probes complementary to a methylation site can be clonedand/or synthesized. Any suitable label can be used with a probe.Detectable labels suitable for use with nucleic acid probes include, forexample, any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels include biotin for staining with labeledstreptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels,enzymes, and colorimetric labels. Other labels include ligands whichbind to antibodies labeled with fluorophores, chemiluminescent agents,and enzymes. A probe can also constitute radiolabelled PCR primers thatare used to generate a radiolabelled amplicon. Labeling strategies forlabeling nucleic acids and corresponding detection strategies can befound, e.g., in Haugland (2003) Handbook of Probes and ResearchChemicals Ninth Edition by Molecular Probes, Inc. (Eugene Oreg.).Additional non-limiting details regarding sequence detection strategiesare found below.

PCR, RT-PCR and LCR are in particularly broad use as amplification andamplification-detection methods for amplifying nucleic acids (e.g.,those comprising a methylation site), facilitating detection of thenucleic acids of interest.

In embodiments, real time PCR or LCR is performed on the amplificationmixtures described herein, e.g., using molecular beacons or TaqMan™probes. A molecular beacon (MB) is an oligonucleotide or peptide nucleicacid (PNA) which, under appropriate hybridization conditions,self-hybridizes to form a stem and loop structure. The MB has a labeland a quencher at the termini of the oligonucleotide or PNA; thus, underconditions that permit intra-molecular hybridization, the label istypically quenched (or at least altered in its fluorescence) by thequencher. Under conditions where the MB does not display intra-molecularhybridization (e.g., when bound to a target nucleic acid, e.g., to aregion of an amplicon during amplification), the MB label is unquenched.Details regarding standard methods of making and using MBs are wellestablished in the literature and MBs are available from a number ofcommercial reagent sources. See also, e.g., Leone et al. (1995)“Molecular beacon probes combined with amplification by NASBA enablehomogenous real-time detection of RNA.” Nucleic Acids Res. 26:2150-2155;Tyagi and Kramer (1996) “Molecular beacons: probes that fluoresce uponhybridization” Nature Biotechnology 14:303-308; Blok and Kramer (1997)“Amplifiable hybridization probes containing a molecular switch” MolCell Probes 11:187-194; Hsuih et al. (1997) “Novel, ligation-dependentPCR assay for detection of hepatitis C in serum” J Clin Microbiol34:501-507; Kostrikis et al. (1998) “Molecular beacons: spectralgenotyping of human alleles” Science 279:1228-1229; Sokol et al. (1998)“Real time detection of DNA:RNA hybridization in living cells” Proc.Natl. Acad. Sci. U.S.A. 95:11538-11543; Tyagi et al. (1998) “Multicolormolecular beacons for allele discrimination” Nature Biotechnology16:49-53; Bonnet et al. (1999) “Thermodynamic basis of the chemicalspecificity of structured DNA probes” Proc. Natl. Acad. Sci. U.S.A.96:6171-6176; Fang et al. (1999) “Designing a novel molecular beacon forsurface-immobilized DNA hybridization studies” J. Am. Chem. Soc.121:2921-2922; Marras et al. (1999) “Multiplex detection ofsingle-nucleotide variation using molecular beacons” Genet. Anal.Biomol. Eng. 14:151-156; and Vet et al. (1999) “Multiplex detection offour pathogenic retroviruses using molecular beacons” Proc. Natl. Acad.Sci. U.S.A. 96:6394-6399. Additional details regarding MB constructionand use is found in the patent literature, e.g., U.S. Pat. No. 5,925,517(Jul. 20, 1999) to Tyagi et al. entitled “Detectably labeled dualconformation oligonucleotide probes, assays and kits;” U.S. Pat. No.6,150,097 to Tyagi et al (Nov. 21, 2000) entitled “Nucleic aciddetection probes having non-FRET fluorescence quenching and kits andassays including such probes” and U.S. Pat. No. 6,037,130 to Tyagi et al(Mar. 14, 2000), entitled “Wavelength-shifting probes and primers andtheir use in assays and kits.”

PCR detection and quantification using dual-labeled fluorogenicoligonucleotide probes, commonly referred to as “TaqMan™” probes, canalso be performed. These probes are composed of short (e.g., 20-25 base)oligodeoxynucleotides that are labeled with two different fluorescentdyes. On the 5′ terminus of each probe is a reporter dye, and on the 3′terminus of each probe a quenching dye is found. The oligonucleotideprobe sequence is complementary to an internal target sequence presentin a PCR amplicon. When the probe is intact, energy transfer occursbetween the two fluorophores and emission from the reporter is quenchedby the quencher by FRET. During the extension phase of PCR, the probe iscleaved by 5′ nuclease activity of the polymerase used in the reaction,thereby releasing the reporter from the oligonucleotide-quencher andproducing an increase in reporter emission intensity. Accordingly,TaqMan™ probes are oligonucleotides that have a label and a quencher,where the label is released during amplification by the exonucleaseaction of the polymerase used in amplification. This provides a realtime measure of amplification during synthesis. A variety of TaqMan™reagents are commercially available, e.g., from Applied Biosystems(Division Headquarters in Foster City, Calif.) as well as from a varietyof specialty vendors such as Biosearch Technologies (e.g., black holequencher probes). Further details regarding dual-label probe strategiescan be found, e.g., in WO92/02638.

Other similar methods include e.g. fluorescence resonance energytransfer between two adjacently hybridized probes, e.g., using the“LightCycler™” format described in U.S. Pat. No. 6,174,670.

Amplification and Sequencing Primers

In embodiments, methylation sites are detected using primers, e.g., toamplify and/or sequence polynucleotides comprising the methylationsites.

Suitable primers can be designed and is not intended that the presentsubject matter be limited to any particular primer or primer pair. Forexample, primers can be designed using any suitable software program,such as LASERGENE™, e.g., taking account of publicly available sequenceinformation. Flanking sequences for the methylation sites identifiedherein are publicly available; accordingly, suitable amplificationprimers can be constructed based on well understood base-pairing rules.The sequence of any amplicon can be detected as has already beendiscussed above, e.g., by sequencing, hybridization, arrayhybridization, PCR, LCR, or the like.

In embodiments, the primers are radiolabelled, or labeled by anysuitable means (e.g., using a non-radioactive fluorescent tag), to allowfor rapid visualization of differently sized amplicons following anamplification reaction without any additional labeling step orvisualization step. In embodiments, the primers are not labeled, and theamplicons are visualized following their size resolution, e.g.,following agarose or acrylamide gel electrophoresis. In embodiments,ethidium bromide staining of the PCR amplicons following size resolutionallows visualization of the different size amplicons.

It is not intended that the primers be limited to generating an ampliconof any particular size. The primers can generate an amplicon of anysuitable length for detection (e.g., by sequencing or hybridization). Inembodiments, amplification produces an amplicon at least 20 nucleotidesin length, or alternatively, at least 50 nucleotides in length, oralternatively, at least 100 nucleotides in length, or alternatively, atleast 200 nucleotides in length. Amplicons of any size can be detectedand/or sequenced using various technologies described herein and knownin the art.

Detection of Methylation Levels Using Sequencing

Sequencing is the process of determining the precise order ofnucleotides within a DNA molecule. The advent of rapid DNA sequencingmethods has greatly accelerated biological and medical research anddiscovery. Non-limiting examples and descriptions are provided below.However, embodiments are not limited to the use of a particularsequencing assay, technology, or approach.

Sanger sequencing is a method of DNA sequencing based on the selectiveincorporation of chain-terminating dideoxynucleotides by DNA polymeraseduring in vitro DNA replication (Sanger F; Coulson A R (May 1975) J.Mol. Biol. 94 (3): 441-8; Sanger et al. (December 1977) Proc. Natl.Acad. Sci. U.S.A. 74 (12): 5463-7).

In embodiments, next-generation sequencing is used. Non-limitingexamples of next-generation sequencing methods include massivelyparallel signature sequencing (MPSS), single-molecule real-timesequencing, ion semiconductor sequencing, pyrosequencing, sequencing bysynthesis, sequencing by ligation, chain termination, DNA nanoballsequencing, helicos single molecule sequencing, single molecule realtime sequencing, nanopore DNA sequencing, tunnelling currents DNAsequencing, and sequencing by hybridization.

Many commercially available sequencing technologies, devices, andservices are available. In embodiments, an Illumina sequencer is used.In embodiments, PCR products are ligated with a linker and sequencedusing a high throughput sequencer, such as an Illumina sequencer. Inembodiments, the ligation step can be avoided, omitted, or eliminated byadding a linker to amplification primers.

Array-Based Sequence Detection

Array-based detection can be performed using commercially availablearrays, e.g., from Affymetrix (Santa Clara, Calif.) or othermanufacturers. Reviews regarding the operation of nucleic acid arraysinclude Sapolsky et al. (1999) “High-throughput polymorphism screeningand genotyping with high-density oligonucleotide arrays.” GeneticAnalysis: Biomolecular Engineering 14:187-192; Lockhart (1998) “Mutantyeast on drugs” Nature Medicine 4:1235-1236; Fodor (1997) “Genes, Chipsand the Human Genome.” FASEB Journal 11:A879; Fodor (1997) “MassivelyParallel Genomics.” Science 277: 393-395; and Chee et al. (1996)“Accessing Genetic Information with High-Density DNA Arrays.” Science274:610-614.

A variety of probe arrays have been described in the literature and canbe used for detection of methylation. For example, DNA probe array chipsor larger DNA probe array wafers (from which individual chips wouldotherwise be obtained by breaking up the wafer) may be used inembodiments described herein. DNA probe array wafers generally compriseglass wafers on which high density arrays of DNA probes (short segmentsof DNA) have been placed. Each of these wafers can hold, for example,approximately 60 million DNA probes that are used to recognize longersample DNA sequences (e.g., from individuals or populations, e.g., thatcomprise methylation sites of interest). The recognition of sample DNAby the set of DNA probes on the glass wafer takes place through DNAhybridization. When a DNA sample hybridizes with an array of DNA probes,the sample binds to those probes that are complementary to the sampleDNA sequence. By evaluating to which probes the sample DNA for anindividual hybridizes more strongly, it is possible to determine whethera known sequence of nucleic acid is present or not in the sample,thereby determining whether a uracil, thymine, or cytosine is present ata polynucleotide site corresponding to a genomic methylation site. Onecan also use this approach to control the hybridization conditions topermit single nucleotide discrimination, e.g., for the identification ofmethylation at a site of interest. Arrays provide one convenientembodiment for detecting multiple methylation sites simultaneously (orin series). Of course, any detection technology (PCR, LCR, and/orsequencing etc.) can similarly be used, e.g., with multiplexamplification/detection/sequencing reactions, or simply by runningseveral separate reactions, e.g., simultaneously or in series.

In embodiments, the use of DNA probe arrays to obtain methylationinformation involves the following general steps: design and manufactureof DNA probe arrays, preparation of the sample, bisulfite treatment,hybridization of sample DNA to the array, detection of hybridizationevents and data analysis to determine sequence. In embodiments, an arrayis used to capture polynucleotides containing a methylation site ofinterest, and the captured polynucleotides are subsequently amplifiedand/or sequenced. Preferred wafers are manufactured using a processadapted from semiconductor manufacturing to achieve cost effectivenessand high quality, and are available, e.g., from Affymetrix, Inc. ofSanta Clara, Calif.

For example, probe arrays can be manufactured by light-directed chemicalsynthesis processes, which combine solid-phase chemical synthesis withphotolithographic fabrication techniques as employed in thesemiconductor industry. Using a series of photolithographic masks todefine chip exposure sites, followed by specific chemical synthesissteps, the process constructs high-density arrays of oligonucleotides,with each probe in a predefined position in the array. Multiple probearrays can be synthesized simultaneously on a large glass wafer. Thisparallel process enhances reproducibility and helps achieve economies ofscale.

In embodiments, DNA probe arrays can be used to obtain data regardingpresence of sequences (e.g., corresponding to methylated or unmethylatedDNA) of interest. The DNA samples may be tagged with biotin and/or afluorescent reporter group by standard biochemical methods. The labeledsamples are incubated with an array, and segments of the samples bind,or hybridize, with complementary sequences on the array. The array canbe washed and/or stained to produce a hybridization pattern. The arrayis then scanned and the patterns of hybridization are detected byemission of light from the fluorescent reporter groups. Because theidentity and position of each probe on the array is known, the nature ofthe DNA sequences in the sample applied to the array can be determined.

In embodiments, the nucleic acid sample to be analyzed is isolated,bisulfite-treated, amplified and, optionally, labeled with biotin and/ora fluorescent reporter group. The labeled nucleic acid sample may thenbe incubated with the array using a fluidics station and hybridizationoven. The array can be washed and or stained or counter-stained, asappropriate to the detection method. After hybridization, washing andstaining, the array is inserted into a scanner, where patterns ofhybridization are detected. The hybridization data are collected aslight emitted from the fluorescent reporter groups already incorporatedinto the labeled nucleic acid, which is now bound to the probe array.Probes that most clearly match the labeled nucleic acid produce strongersignals than those that have mismatches. Since the sequence and positionof each probe on the array are known, by complementarity, the identityof the nucleic acid sample applied to the probe array can be identified.In embodiments, hybridization techniques and conditions that allow onlyfully complementary nucleotide sequences to hybridize with probes in anarray are used.

Prior to amplification and/or detection of a nucleic acid comprising asequence of interest, the nucleic acid is optionally purified from thesamples by any available method, e.g., those taught in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001 (“Sambrook”);and/or Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2002) (“Ausubel”)). A plethora of kits are also commercially availablefor the purification of nucleic acids from cells or other samples (see,e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean™,from Stratagene; and, QIAprep™ from Qiagen). Alternately, samples cansimply be directly subjected to amplification or detection, e.g.,following aliquotting and/or dilution.

Breast Cancer Diagnostic System and Processes

In embodiments, included herein is a system for detecting methylation orunmethylation of a DCIS cancer cell proliferation DNA molecule of asubject. In embodiments, the system provides at least one processor; andat least one memory including program code which when executed by the atleast one processor provides operations comprising: contacting anisolated DCIS cancer cell proliferation DNA molecule from the subjectwith a bisulfite salt thereby forming a reacted DCIS cancer cellproliferation DNA molecule; detecting the presence or absence of uracilin the reacted DCIS cancer cell proliferation DNA molecule at amethylation site set forth in Table 1, thereby detecting methylation orunmethylation of the DCIS cancer cell proliferation DNA molecule of thesubject; generating a diagnosis for the subject based at least in parton the presence or absence of uracil in the reacted DCIS cancer cellproliferation DNA molecule at the methylation site set forth in Table 1;and providing, via a user interface, the diagnosis or prognosis for thesubject. In embodiments, the system provides at least one processor; andat least one memory including program code which when executed by the atleast one processor provides operations comprising: contacting theplurality of isolated DCIS cancer cell proliferation DNA molecules witha bisulfite salt thereby forming a plurality of reacted DCIS cancer cellproliferation DNA molecules; detecting the level of reacted DCIS cancercell proliferation DNA molecules in the plurality of reacted DCIS cancercell proliferation DNA molecules having a uracil at a methylation siteset forth in Table 1 thereby detecting the level of methylation orunmethylation in the plurality of DCIS cancer cell proliferation DNAmolecules of the subject; generating a diagnosis for the subject basedat least in part on the level of methylation or unmethylation at theplurality of methylation sites set forth in Table 1; and providing, viaa user interface, the diagnosis or prognosis for the subject.

FIG. 2 depicts a block diagram illustrating an exemplary breast cancerdiagnostic system 600. Referring to FIG. 2, the breast cancer diagnosticsystem 600 can include an input module 610, an isolation module 612, aconversion module 614, a detection module 616, a diagnosis module 618, atreatment module 620, and a user interface (UI) module 622. The breastcancer diagnostic system 600 can be configured to provide a diagnosisindicative of a presence of IDC and/or a risk of developing IDC.Moreover, the breast cancer diagnostic system 600 can be furtherconfigured to generate a treatment plan for a subject based on thediagnosis. For instance, when the diagnosis indicates a presence and/orrisk of IDC in a subject, the breast cancer diagnostic system 600 canrecommend one or more treatments including, for example, surgery (e.g.,lumpectomy or mastectomy), radiation therapy, chemotherapy, hormonetherapy, targeted therapy, and/or administration of an active agent.

One or more modules of the breast cancer diagnostic system 600 can berealized in digital electronic circuitry, integrated circuitry,specially designed application specific integrated circuits (ASICs),field programmable gate arrays (FPGAs) computer hardware, firmware,software, and/or combinations thereof. The breast cancer diagnosticsystem 600 can further be communicatively coupled with one or moredevices including, for example, a device 630. The breast cancerdiagnostic system 600 can communicate with the device 620 via a wiredand/or wireless network 640 (e.g., a wide area network (WAN), a localarea network (LAN), and/or the Internet). As shown in FIG. 2, the breastcancer diagnostic system 600 can be further coupled with a data store650.

The input module 610 can be adapted to receive and/or collect a sampleof a DCIS cancer cell proliferation obtained from a subject. Theisolation module 612 can be configured to isolate DNA from the DCIScancer cell proliferation sample received by the input module 610thereby forming isolated DCIS cancer cell proliferation DNA. Theconversion module 614 can be configured to treat the isolated DCIScancer cell proliferation DNA including by contacting the isolated DCIScancer cell proliferation DNA with one or more bisulfite reagentsincluding, for example, a bisulfite salt. Exposing the isolated DCIScancer cell proliferation DNA to one or more bisulfite reagents canconvert cytosine to uracil while 5-mC is left unmodified. Thus, the 5-mCpresent in the isolated DCIS cancer cell proliferation DNA will remainin the reacted DCIS cancer cell proliferation DNA. Meanwhile, anycytosine in the isolated DCIS cancer cell proliferation DNA will bereplaced by uracil in the reacted DCIS cancer cell proliferation DNA. Inembodiments, the treatment of the isolated DCIS cancer cellproliferation DNA can be performed by applying one or more kits (e.g.,the Bisulflash DNA Modification Kit (Epigentek) or Imprint DNAModification Kit (Sigma)).

In embodiments, the conversion module 614 can be further adapted toensure optimal bisulfite conversion (e.g., with desired DNA fragmentsize for post-bisulfite ligation) by controlling one or more of aconcentration of the bisulfite reagents, temperature, and reaction timeperiod. It should be appreciated that the conversion module 614 can beadapted to use a different and/or additional type of reagent withoutdeparting from the scope of the present subject matter. For example, theconversion module 614 can treat the isolated DCIS cancer cellproliferation DNA with potassium chloride, which may reduce thethermophilic DNA degradation associated with the conversion of cytosineto uracil. Moreover, the conversion module 614 can be configured toperform additional processing of the reacted DCIS cancer cellproliferation DNA including, for example, desulphonation (e.g., with analkalized solution), cleansing (e.g., by elution), and amplification(e.g., using the PCR method).

The detection module 616 can be configured to detect a methylationand/or unmethylation of the DCIS cancer cell proliferation DNA. Forinstance, the detection module 616 can detect methylation by detecting apresence of uracil in the reacted DCIS cancer cell proliferation DNAgenerated by the conversion module 614. Alternately and/or additionally,the detection module 616 can detect unmethylation by detecting anabsence of uracil in the reacted DCIS cancer cell proliferation DNA. Inembodiments, the detection module 616 can be configured detect thepresence and/or absence of uracil at specific methylation sites. Thatis, the detection module 616 can be configured to detect the presenceand/or absence of uracil at specific chromosomal positions of certainchromosomes. For example, the breast cancer diagnostic system 600 canstore a plurality of specific methylation sites (e.g., Table 1) in thedata store 650. As such, to detect methylation, the detection module 616can be configured to obtain, from the data store 650, one or morespecific methylation sites at which to test for the presence and/orabsence of uracil. Moreover, in embodiments, the detection module 616can be configured to determine a level of methylation and/orunmethylation at the specific methylation sites. The level ofmethylation at a particular site can correspond to a proportion of thereacted DCIS cancer cell proliferation DNA that has a cytosine ratherthan a uracil at that site. By contrast, the level of unmethylation at aparticular site can correspond to a proportion of reacted DCIS cancercell proliferation DNA that has a uracil rather than a cytosine at thatsite.

In embodiments, the conversion module 614 may amplify the reacted DCIScancer cell proliferation DNA such as by using a PCR method. Thedetection of methylation and/or unmethylation in amplified reacted DCIScancer cell proliferation DNA may require detection of a presence and/orabsence of thymidine at a site of interest in amplicons amplified fromthe reacted DCIS cancer cell proliferation DNA. That is, instead ofdetecting the presence and/or absence of uracil, the detection module616 can be configured to detect methylation and/or unmethylation ofamplified reacted DCIS cancer cell proliferation DNA by detecting apresence and/or absence of thymidine at specific methylation sites(e.g., as set forth in Table 1).

The diagnosis module 618 can be configured to generate a diagnosis forthe subject based on whether the detection module 616 detectsmethylation and/or unmethylation at the plurality of specificmethylation sites (e.g., Table 1). Alternately or additionally, thediagnosis module 618 can be configured to generate a diagnosis for thesubject based on a level of methylation and/or unmethylation detected bythe detection module 616 at the plurality of specific methylation sites.For instance, diagnosis module 618 can determine that the DCIS cancercell proliferation is invasive competent when the unmethylation level(e.g., proportion of uracil) at different methylation sites exceeds thecorresponding thresholds (e.g., as set forth in Table 2).

The treatment module 620 can be configured to formulate a treatment planfor the subject based on the diagnosis generated by the diagnosis module618. For instance, when the diagnosis generated by the diagnosis module618 indicates a presence and/or risk of IDC, the treatment module 620can prescribe or suggest one or more treatments including, for example,surgery, radiation therapy, chemotherapy, hormone therapy, and/oradministration of an active agent. In embodiments, the treatment module620 can be configured to provide the treatment plan to the device 630via the network 640. Alternately or additionally, the treatment module620 can store the treatment plan in the data store 650.

The UI module 622 can be configured to generate a UI through which auser (e.g., a physician) can interface with the breast cancer diagnosticsystem 600. For example, the UI module 622 can provide one or moregraphic user interfaces (GUIs) configured to display the diagnosisand/or treatment plan for the subject.

FIG. 5 depicts a flowchart illustrating an exemplary process 700 fordiagnosing invasive or invasive competent breast cancer. Referring toFIGS. 3 and 4, the process 700 can be performed by the breast cancerdiagnostic system 600.

The breast cancer diagnostic system 600 (e.g., the input module 610) canreceive a sample of a DCIS cancer cell proliferation from a subject(702). The breast cancer diagnostic system 600 (e.g., the isolationmodule 612) can isolate DCIS cancer cell proliferation DNA from the DCIScancer cell proliferation sample (704). The breast cancer diagnosticsystem 600 (e.g., the conversion module 614) can treat the isolated DCIScancer cell proliferation DNA with a bisulfite salt to generate reactedDCIS cancer cell proliferation DNA (706). Treating the isolated DCIScancer cell proliferation DNA with the bisulfite salt can form a reactedDCIS cancer cell proliferation DNA by converting the cytosine present inthe isolated DCIS cancer cell proliferation DNA to uracil. Inembodiments, the breast cancer diagnostic system 600 can further processthe reacted DCIS cancer cell proliferation DNA by desulphonating,cleansing, and/or amplifying the reacted DCIS cancer cell proliferationDNA.

The breast cancer diagnostic system 600 (e.g., the detection module 616)can detect methylation and/or unmethylation of the isolated DCIS cancercell proliferation DNA by at least detecting a presence and/or absenceof uracil in the reacted DCIS cancer cell proliferation DNA (708). Inembodiments, the breast cancer diagnostic system 600 can be configuredto detect a presence and/or absence of uracil at specific methylationsites (e.g., as set forth in Table 1). Moreover, the breast cancerdiagnostic system 600 can be configured to detect a level of methylationand/or unmethylation at the methylation sites.

The breast cancer diagnostic system 600 (e.g., the diagnostics module618) can generate a diagnosis for the subject based on the methylationand/or unmethylation of the isolated DCIS cancer cell proliferation DNA(710). For example, the breast cancer diagnostic system 600 can generatea diagnosis based on a level of methylation and/or unmethylation at aplurality of specific methylation sites. Each methylation site may beassociated with a certain threshold unmethylation level (e.g., as setforth in Table 2). As such, the breast cancer diagnostic system 600 candetermine that the DCIS cancer cell proliferation from the subject isinvasive if the level of unmethylation at the plurality of methylationsites exceeds (e.g., as set forth in Table 2) or is below (e.g., as setforth in Table 3) the corresponding thresholds.

The breast cancer diagnostic system 600 (e.g., the treatment module 620)can formulate, based on the diagnosis, a treatment plan for the subject(712). For example, when the diagnosis indicates that a presence and/orrisk of IDC in the subject, the breast cancer diagnostic system 600 canprescribe or suggest surgery, radiation therapy, chemotherapy, hormonetherapy, and/or administration of an active agent. The breast cancerdiagnostic system 600 (e.g., the UI module 622) can provide, via a UI(e.g., GUI at the device 630), the diagnosis and/or the treatment planfor the subject (714).

FIG. 6 depicts a flowchart illustrating an exemplary process 800 fordiagnosing IDC. Referring to FIGS. 2 and 4, the process 700 can beperformed by the breast cancer diagnostic system 600.

The breast cancer diagnostic system 600 (e.g., the input module 610) canreceive a sample of a DCIS cancer cell proliferation from a subject(802). The breast cancer diagnostic system 600 (e.g., the isolationmodule 612) can isolate DCIS cancer cell proliferation DNA from the DCIScancer cell proliferation sample (804). The breast cancer diagnosticsystem 600 (e.g., the conversion module 614) can treat the isolated DCIScancer cell proliferation DNA with a bisulfite salt to generate reactedDCIS cancer cell proliferation DNA (806).

As shown in FIG. 4, the breast cancer diagnostic system 600 (e.g., theconversion module 614) can amplify the reacted DCIS cancer cellproliferation DNA (808). For instance, the breast cancer diagnosticsystem 600 can amplify the reacted DCIS cancer cell proliferation DNAsubsequent to treating the isolated DCIS cancer cell proliferation DNAwith the bisulfite salt to generate amplicons of the reacted DCIS cancercell proliferation DNA. The breast cancer diagnostic system 600 candetect methylation and/or unmethylation of the isolated DCIS cancer cellproliferation DNA by detecting a presence and/or absence of thymidine inthe amplified reacted DCIS cancer cell proliferation DNA (810).

The breast cancer diagnostic system 600 (e.g., the diagnostics module618) can generate a diagnosis for the subject based on the methylationand/or unmethylation of the isolated DCIS cancer cell proliferation DNA(812). Moreover, the breast cancer diagnostic system 600 (e.g., thetreatment module 620) can formulate, based on the diagnosis, a treatmentplan for the subject (814). The breast cancer diagnostic system 600(e.g., the UI module 622) can provide, via a UI, the diagnosis and/ortreatment plan for the subject.

It should be appreciated that the process 700 and/or 800 can includedifferent and/or additional operations without departing from the scopeof the present subject matter. Moreover, one or more operations of theprocess 700 and/or 800 can be omitted and/or repeated without departingfrom the scope of the present subject matter.

Implementations of the present subject matter can include, but are notlimited to, methods consistent with the descriptions provided above aswell as articles that comprise a tangibly embodied machine-readablemedium operable to cause one or more machines (e.g., computers, etc.) toresult in operations implementing one or more of the described features.Similarly, computer systems are also described that can include one ormore processors and one or more memories coupled to the one or moreprocessors. A memory, which can include a computer-readable storagemedium, can include, encode, store, or the like one or more programsthat cause one or more processors to perform one or more of theoperations described herein. Computer implemented methods consistentwith one or more implementations of the current subject matter can beimplemented by one or more data processors residing in a singlecomputing system or multiple computing systems. Such multiple computingsystems can be connected and can exchange data and/or commands or otherinstructions or the like via one or more connections, including but notlimited to a connection over a network (e.g. the Internet, a wirelesswide area network, a local area network, a wide area network, a wirednetwork, or the like), via a direct connection between one or more ofthe multiple computing systems, etc.

One or more aspects or features of the subject matter described hereincan be realized in digital electronic circuitry, integrated circuitry,specially designed ASICs, FPGAs, computer hardware, firmware, software,and/or combinations thereof. These various aspects or features caninclude implementation in one or more computer programs that areexecutable and/or interpretable on a programmable system including atleast one programmable processor, which can be special or generalpurpose, coupled to receive data and instructions from, and to transmitdata and instructions to, a storage system, at least one input device,and at least one output device. The programmable system or computingsystem can include clients and servers. A client and server aregenerally remote from each other and typically interact through acommunication network. The relationship of client and server arises byvirtue of computer programs running on the respective computers andhaving a client-server relationship to each other.

These computer programs, which can also be referred to programs,software, software applications, applications, components, or code,include machine instructions for a programmable processor, and can beimplemented in a high-level procedural language, an object-orientedprogramming language, a functional programming language, a logicalprogramming language, and/or in assembly/machine language. As usedherein, the term “machine-readable medium” refers to any computerprogram product, apparatus and/or device, such as for example magneticdiscs, optical disks, memory, and Programmable Logic Devices (PLDs),used to provide machine instructions and/or data to a programmableprocessor, including a machine-readable medium that receives machineinstructions as a machine-readable signal. The term “machine-readablesignal” refers to any signal used to provide machine instructions and/ordata to a programmable processor. The machine-readable medium can storesuch machine instructions non-transitorily, such as for example as woulda non-transient solid-state memory or a magnetic hard drive or anyequivalent storage medium. The machine-readable medium can alternativelyor additionally store such machine instructions in a transient manner,such as for example as would a processor cache or other random accessmemory associated with one or more physical processor cores.

To provide for interaction with a user, one or more aspects or featuresof the subject matter described herein can be implemented on a computerhaving a display device, such as for example a cathode ray tube (CRT) ora liquid crystal display (LCD) or a light emitting diode (LED) monitorfor displaying information to the user and a keyboard and a pointingdevice, such as for example a mouse or a trackball, by which the usermay provide input to the computer. Other kinds of devices can be used toprovide for interaction with a user as well. For example, feedbackprovided to the user can be any form of sensory feedback, such as forexample visual feedback, auditory feedback, or tactile feedback; andinput from the user can be received in any form, including, but notlimited to, acoustic, speech, or tactile input. Other possible inputdevices include, but are not limited to, touch screens or othertouch-sensitive devices such as single or multi-point resistive orcapacitive trackpads, voice recognition hardware and software, opticalscanners, optical pointers, digital MRI image capture devices andassociated interpretation software, and the like.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

Example 1. Development of an Epigenetic Biomarker Panel for DCISInvasiveness

DNA methylation patterns were evaluated in 7 DCIS samples from patientswithout reported invasion (pure DCIS), and 3 DCIS samples from patientswith IDC. In order to identify DNA methylation changes exclusive tocancer cells, malignant cells located within the involved ducts of DICSwere isolated by laser capture microdissection (FIG. 1A). Genome-wideprofiling of DNA methylation was performed. Reduced representationbisulfite sequencing (RRBS) was used to evaluate DNA methylationpatterns at gene promoters in captured cells (FIG. 1C). Clusteringanalysis clearly separated DCIS samples into two epigenetic groups. Thefirst DCIS epigenetic group was associated with almost no DNAmethylation at promoters and obtained from patients lacking invasion(pure DCIS). At the same time, specimens from the second epigeneticgroup of DCIS were characterized by extensive accumulation of DNAmethylation at promoters. This group contained all analyzed DCIS frompatients with IDC and 3 pure DCIS, according to a clinical pathologyreport.

In general, DNA methylation patterns strikingly separate normal fromcancer cells and reflect genome-wide alterations of chromatin. Whilenormal cells are missing DNA methylation at promoters, cancer ischaracterized by aberrant accumulation of promoter DNA methylation,which is frequently associated with gene silencing. Therefore, the firstDCIS epigenetic group has a DNA methylation pattern very similar tonormal cells. At the same time, the second DCIS epigenetic groupexhibited a clear accumulation of cancer-associated DNA methylation andwas highly enriched in DCIS associated with invasion.

Not to be bound by scientific theory, it was hypothesized that there aretwo different epigenetic programs driving DCIS progression, and patientswith the “invasion incompetent” signature are unlikely to developinvasion while patients with pure DCIS from the second epigenetic grouphaving “invasion competent” signature will have a very high chance todevelop invasion. This hypothesis on the predictive power of epigeneticprofiling is supported by the fact that on independent blinded pathologyreview of the “pure DCIS” cases, DCIS sample number 6 (DCIS_6) wasreclassified as invasive (by a pathologist) that confirms the “invasioncompetent” signature identified by DNA methylation analysis. Thus, basedon the DNA methylation pattern, the potential for invasiveness of DCIScan be predicted.

140 cytosines with DNA methylation patterns that strictly distinguishthese two DCIS groups (“invasion incompetent” and “invasion competent”)were identified (FIG. 1D). These epigenetic signatures can be used indiagnostic and prognostic DCIS tests for invasiveness that guideclinical management and potentially de-escalate therapy for DCIS with nopotential for invasion.

DNA Methylation Profiling

By using LDM 7000 (Leica Microsystems), cancer cells were isolated frombreast tissues by using laser capture procedure. Genomic DNA waspurified by using a standard phenol/chloroform extraction approachfollowed by ethanol precipitation. Further genomic DNA underwent RRBSprocedure. RRBS DNA amplicons were paired-end sequenced by using HighSeq(Illumina). For each sample, at least 15 million aligned reads wereobtained. Specific methylation signatures for “invasion incompetent” and“invasion competent” cells were determined based on cytosines which arecharacterized by at least 5 sequencing reads in each sample.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of detecting methylation or unmethylation of a ductalcarcinoma in situ (DCIS) cell proliferation deoxyribonucleic acid (DNA)molecule of a human subject, the method comprising: (i) contacting anisolated DCIS cancer cell proliferation DNA molecule from a breasttissue sample of said subject with a bisulfate salt thereby forming areacted DCIS cancer cell proliferation DNA molecule; and (ii) detectingthe presence or absence of uracil in said reacted DCIS cancer cellproliferation DNA molecule at a methylation site, wherein saidmethylation site is at Chromosome 1 (Chr1) position 4714314, Chromosome3 (Chr3) position 121903470, Chromosome 4 (Chr4) position 44449864,Chromosome 7 (Chr7) position 157477232, Chromosome 12 (Chr12) position95941925, Chr12 position 129338355, Chromosome 19 (Chr19) 30017283, orChromosome 20 (Chr20) position 23016002 with respect to human genomeassembly hg19, thereby detecting methylation or unmethylation of saidDCIS cancer cell proliferation DNA molecule of said subject. 2.(canceled)
 3. The method of claim 1, further comprising detecting thepresence or absence of uracil in a plurality of reacted DCIS cancer cellproliferation DNA molecules at a plurality of methylation sites selectedfrom Chr1 position 4714314, Chr1 position 11413742, Chr1 position39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1position 207669851, Chr1 position 237205704, Chr1 position 237205705,Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2)position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2position 115919663, Chr2 position 115920004, Chr2 position 118982006,Chr2 position 177001540, Chr3 position 14852857, Chr3 position121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3position 170303423, Chr3 position 170303424, Chr3 position 170303425,Chr 4 position 44449864, Chr4 position 54976099, Chr4 position 56023880,Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5position 87981177, Chr5 position 140743998, Chr5 position 178421786,Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6position 157557787, Chr6 position 160769248, Chr7 position 1282082, Chr7position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7position 100946148, Chr7 position 100946151, Chr7 position 121957003,Chr7 position 150038502, Chr7 position 157477232, Chr7 position157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10position 123778639, Chr10 position 123778640, Chr10 position 124902829,Chr10 position 124909545, Chr10 position 130085373, Chr10 position134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position66187593, Chr11 position 71318977, Chr11 position 101453451, Chr12position 49726711, Chr12 position 50297756, Chr12 position 50297763,Chr12 position 50297768, Chr12 position 50297774, Chr12 position50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12position 95941925, Chr12 position 128750309, Chr 12 position 129338355,Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17(Chr17) position 3211643, Chr17 position 30244229, Chr17 position35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18position 19745054, Chr18 position 19747206, Chr18 position 44774403,Chr18 position 55103840, Chr18 position 55106910, Chr18 position70534832, Chr18 position 72880039, Chr18 position 77547934, Chr19position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chr20 position 1294019, Chr20 position 3073503,Chr20 position 10198305, Chr20 position 23015989, Chr20 position23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20position 53092916, Chr20 position 59827619, Chr20 position 59828325,Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21position 9826934, or Chromosome 22 position 43807517 with respect tohuman genome assembly hg19.
 4. (canceled)
 5. (canceled)
 6. The method ofclaim 3, wherein said plurality of methylation sites comprises at leastabout 2, 5, or 10 methylation sites with respect to human genomeassembly hg19.
 7. The method of claim 3, wherein the uracil level of atleast one methylation site of said plurality of methylation sites isabove a threshold selected from 76.09 for chr1 position 11413742, 78.57for chr1 240934954, 74.49 for chr10 position 123778639, 79.31 for chr10position 123778640, 74.47 for chr11 position 1215978, or 83.33 for chr12position 128750309 with respect to human genome assembly hg19.
 8. Themethod of claim 3, wherein the uracil level of at least one methylationsite of said plurality of methylation sites is below a thresholdselected from 74.32 for chr1 position 4714314, 63.81 for chr1 position39957798, 85.11 for chr1 46951513, 73.08 for chr1 position 47904912,71.20 for chr1 position 62660691, 46.94 for chr1 position 63785800,42.86 for chr1 position 67600465, 48.28 for chr1 position 91183172,64.29 for chr1 position 166853786, 88.89 for chr1 position 179545096,89.29 for chr1 position 207669851, 82.61 for chr1 position 237205704,78.57 for chr1 position 237205705, 60.00 for chr1 position 240161215,75.00 for chr10 position 8076277, 76.30 for chr10 position 50818610,50.43 for chr10 position 77157527, 67.35 for chr10 position 124902829,80.00 for chr10 position 124909545, 76.74 for chr10 position 130085373,34.43 for chr10 position 134598235, 79.31 for chr10 position 134599080,65.85 for chr11 position 9025912, 51.72 for chr11 position 15963013,80.95 for chr11 position 66187593, 51.52 for chr11 position 71318977,57.89 for chr11 position 101453451, 60.00 for chr12 position 49726711,69.81 for chr12 position 50297756, 82.98 for chr12 position 50297763,81.13 for chr12 position 50297768, 75.47 for chr12 position 50297774,77.36 for chr12 position 50297776, 54.17 for chr12 position 50444766,52.00 for chr12 position 75601447, 36.84 for chr12 position 95941925,57.14 for chr12 position 129338355, 60.71 for chr12 position 129338471,65.85 for chr13 position 28502190, 79.84 for chr13 position 79181509,70.70 for chr13 position 92051154, 73.91 for chr13 position 95363553,78.95 for chr13 position 95363592, 72.36 for chr14 position 29236052,62.60 for chr14 position 29236065, 92.45 for chr14 position 101543886,69.84 for chr15 position 29407958, 73.68 for chr15 position 45403826,62.30 for chr15 position 76630094, 39.68 for chr15 position 89951787,71.43 for chr17 position 35294171, 56.52 for chr17 position 64831307,43.48 for chr17 position 74136562, 62.79 for chr17 position 74865566,67.01 for chr18 position 19745047, 64.10 for chr18 position 19745054,80.00 for chr18 position 19747206, 68.29 for chr18 position 44774403,66.20 for chr18 position 55103840, 50.00 for chr18 position 55106910,66.67 for chr18 position 70534832, 33.33 for chr18 position 77547934,38.89 for chr19 position 30016170, 60.00 for chr19 position 30017283,54.24 for chr19 position 30717013, 66.67 for chr19 position 30719659,12.20 for chr2 position 20870821, 34.25 for chr2 position 45156764,34.69 for chr2 position 74743346, 28.21 for chr2 position 80549703,63.64 for chr2 position 105471544, 64.44 for chr2 position 115919663,79.31 for chr2 position 115920004, 47.06 for chr2 position 118982006,63.16 for chr2 position 177001540, 55.43 for chr20 position 1294019,62.96 for chr20 position 3073503, 62.44 for chr20 position 10198305,80.70 for chr20 position 23015989, 65.59 for chr20 position 23016002,55.56 for chr20 position 26189258, 36.00 for chr20 position 48626669,72.13 for chr20 position 53092916, 58.82 for chr20 position 59827619,76.47 for chr20 position 59828325, 37.32 for chr21 position 9825842,50.00 for chr21 position 9826150, 53.36 for chr21 position 9826934,40.68 for chr22 position 43807517, 57.54 for chr3 position 14852857,71.96 for chr3 position 121903470, 68.63 for chr3 position 170303393,67.65 for chr3 position 170303422, 81.82 for chr3 position 170303423,56.52 for chr3 position 170303424, 69.77 for chr3 position 170303425,78.38 for chr4 position 44449864, 72.00 for chr4 position 54976099,55.10 for chr4 position 56023880, 65.00 for chr5 position 71014951,80.82 for chr5 position 72677229, 55.26 for chr5 position 87981177,71.83 for chr5 position 140743998, 56.25 for chr5 position 178421786,57.25 for chr6 position 41337153, 68.18 for chr6 position 85484102,55.56 for chr6 position 157557787, 75.51 for chr6 position 160769248,70.45 for chr7 position 1282082, 31.03 for chr7 position 32467637, 63.89for chr7 position 71801896, 63.89 for chr7 position 71801905, 55.17 forchr7 position 100946148, 43.66 for chr7 position 100946151, 75.00 forchr7 position 121957003, 84.83 for chr7 position 150038502, 26.45 forchr7 position 157477232, 71.43 for chr7 position 157477399, 92.59 forchr7 position 157477401, 70.91 for chr8 position 9764011, 85.51 for chr8position 11566080, 65.22 for chr8 position 11566102, 65.22 for chr8position 11566125, 53.85 for chr8 position 56015232, 95.00 for chr8position 65281933, 44.93 for chr8 position 145105472, 85.19 for chr9position 126780185, 66.96 for chr9 position 127239956, or 84.62 for chr9position 140772369 with respect to human genome assembly hg19.
 9. Themethod of claim 1, wherein said DCIS cancer cell proliferation comprisescancer cells isolated from a sample obtained by biopsy, by laser capturemicrodissection, or by surgical resection of DCIS tissue from saidsubject.
 10. (canceled)
 11. The method of claim 1, wherein said subjecthas undergone lumpectomy, mastectomy, radiation therapy, and/oradministration of an active agent.
 12. The method of claim 11, whereinsaid active agent comprises trastuzumab, trastuzumab emtansine,lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole,letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel,paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib,capecitabine, ixabepilone, methotrexate, or fluorouracil.
 13. (canceled)14. (canceled)
 15. The method of claim 1, wherein said subject (a) is awoman; (b) is about 30 to about 75 years old; (c) has at least onemutant breast cancer 1 (BRCA1), breast cancer 2 (BRCA2), Partner andlocalizer of BRCA2 (PALB2), phosphatase and tensin homolog (PTEN), orp53 allele; (d) has a parent, sibling, or child who has been diagnosedwith breast cancer; (e) has had atypical ductal hyperplasia or lobularcarcinoma in situ; (f) has had previous radiation treatment to the chestor a breast before the age of 30; (g) has received a combination hormonetherapy with estrogen and progestin for at least five years; and/or (h)has or has had breast cancer.
 16. (canceled)
 17. (canceled) 18.(canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled) 27.(canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)32. A deoxyribonucleic acid at least 5 to 100 nucleotides in lengthcomprising a uracil-containing sequence that is identical to a sequenceof at least a 5 contiguous nucleotides within a sequence chosen from SEQID NO:1 to SEQ ID NO:242.
 33. The deoxyribonucleic acid of claim 32,comprising a uracil-containing sequence that is identical to a sequenceof at least 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80,80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160,160-170, 170-180, 180-190, or 190-200 contiguous nucleotides within saidsequence chosen from SEQ ID NO:1 to SEQ ID NO:242.
 34. Thedeoxyribonucleic acid of claim 32, wherein said sequence comprises amethylation site set forth in Table 1 selected from Chr1 position4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1position 237205704, Chr1 position 237205705, Chr1 position 240161215,Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2position 95989474, Chr2 position 105471544, Chr2 position 115919663,Chr2 position 115920004, Chr2 position 118982006, Chr2 position177001540, Chr3 position 14852857, Chr3 position 121903470, Chr3position 170303393, Chr3 position 170303422, Chr3 position 170303423,Chr3 position 170303424, Chr3 position 170303425, Chr 4 position44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5(Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177,Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6)position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6position 160769248, Chr7 position 1282082, Chr7 position 32467637, Chr7position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7position 100946151, Chr7 position 121957003, Chr7 position 150038502,Chr7 position 157477232, Chr7 position 157477399, Chr7 position157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080,Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232,Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9)position 126780185, Chr9 position 127239956, Chr9 position 140772369,Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10position 77157527, Chr10 position 123778639, Chr10 position 123778640,Chr10 position 124902829, Chr10 position 124909545, Chr10 position130085373, Chr10 position 134598235, Chr10 position 134599080,Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11position 15963013, Chr11 position 66187593, Chr11 position 71318977,Chr11 position 101453451, Chr12 position 49726711, Chr12 position50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12position 50297774, Chr12 position 50297776, Chr12 position 50444766,Chr12 position 75601447, Chr12 position 95941925, Chr12 position128750309, Chr 12 position 129338355, Chr12 position 129338471,Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13position 92051154, Chr13 position 95363553, Chr13 position 95363592,Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15position 45403826, Chr15 position 76630094, Chr15 position 89951787,Chromosome 16 position 1255253, Chromosome 17 (Chr17) position 3211643,Chr17 position 30244229, Chr17 position 35294171, Chr17 position64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18position 55106910, Chr18 position 70534832, Chr18 position 72880039,Chr18 position 77547934, Chr19 position 30016170, Chr19 position30017283, Chr19 position 30717013, Chr19 position 30719659, Chr20position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20position 23015989, Chr20 position 23016002, Chr20 position 26189258,Chr20 position 48626669, Chr20 position 53092916, Chr20 position59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position9825842, Chr21 position 9826150, Chr21 position 9826934, or Chromosome22 position 43807517 with respect to human genome assembly hg19.
 35. Thedeoxyribonucleic acid of claim 34, wherein a plurality of saidmethylation sites set forth in Table 1 contain a uracil or a cytosine.36. A deoxyribonucleic acid chosen from SEQ ID NO:243 to SEQ ID NO:356,wherein said nucleic acid is hybridized to a complementary DNA sequencecomprising uridine or cytosine.
 37. The deoxyribonucleic acid of claim36, further comprising an enzyme in a complex with said hybridizedcomplementary DNA sequence.
 38. The deoxyribonucleic acid of claim 37,wherein said enzyme is Taq polymerase.
 39. A kit comprising a pluralityof nucleic acids each independently comprising SEQ ID NO: 242 to SEQ IDNO:356, wherein each nucleic acid of said plurality is unique.
 40. Thekit according to claim 39, further comprising: an enzyme, a reagent fordeamination of cytosine, a buffer, a vial, a control DNA, a device forcollecting a breast tissue sample, device for purification cancer cellsfrom breast tissues, a reagent for isolating DNA, a reagent for labelingDNA, or any combination thereof.
 41. The kit according to claim 40,wherein the enzyme comprises a thermostable DNA polymerase enzyme and/ora restriction enzyme.
 42. A system for detecting methylation orunmethylation of a ductal carcinoma in situ (DCIS) cell massdeoxyribonucleic acid (DNA) molecule of a human subject, the systemcomprising: at least one processor; and at least one memory includingprogram code which when executed by the at least one processor providesoperations comprising: contacting an isolated DCIS cancer cellproliferation DNA molecule from a breast tissue sample of said subjectwith a bisulfite salt thereby forming a reacted DCIS cancer cellproliferation DNA molecule; detecting the presence or absence of uracilin said reacted DCIS cancer cell proliferation DNA molecule at amethylation site set forth in Table 1, wherein said methylation site isat Chromosome 1 (Chr1) position 4714314, Chromosome 3 (Chr3) position121903470, Chromosome 4 (Chr4) position 44449864, Chromosome 7 (Chr7)position 157477232, Chromosome 12 (Chr12) position 95941925, Chr12position 129338355, Chromosome 19 (Chr19) 30017283, or Chromosome 20(Chr20) position 23016002 with respect to human genome assembly hg19,thereby detecting methylation or unmethylation of said DCIS cancer cellproliferation DNA molecule of said subject; generating a diagnosis forsaid subject based at least in part on the presence or absence of uracilin said reacted DCIS cancer cell proliferation DNA molecule at themethylation site; and providing, via a user interface, the diagnosis orprognosis for said subject.
 43. (canceled)
 44. The system of claim 42,wherein the system is further configured to detect the presence orabsence of uracil in a plurality of reacted DCIS cancer cellproliferation DNA molecules at a plurality of methylation sites selectedfrom Chr1 position 4714314, Chr1 position 11413742, Chr1 position39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1position 207669851, Chr1 position 237205704, Chr1 position 237205705,Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2)position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2position 115919663, Chr2 position 115920004, Chr2 position 118982006,Chr2 position 177001540, Chr3 position 14852857, Chr3 position121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3position 170303423, Chr3 position 170303424, Chr3 position 170303425,Chr 4 position 44449864, Chr4 position 54976099, Chr4 position 56023880,Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5position 87981177, Chr5 position 140743998, Chr5 position 178421786,Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6position 157557787, Chr6 position 160769248, Chr7 position 1282082, Chr7position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7position 100946148, Chr7 position 100946151, Chr7 position 121957003,Chr7 position 150038502, Chr7 position 157477232, Chr7 position157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10position 123778639, Chr10 position 123778640, Chr10 position 124902829,Chr10 position 124909545, Chr10 position 130085373, Chr10 position134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position66187593, Chr11 position 71318977, Chr11 position 101453451, Chr12position 49726711, Chr12 position 50297756, Chr12 position 50297763,Chr12 position 50297768, Chr12 position 50297774, Chr12 position50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12position 95941925, Chr12 position 128750309, Chr 12 position 129338355,Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13position 79181509, Chr13 position 92051154, Chr13 position 95363553,Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15)position 29407958, Chr15 position 45403826, Chr15 position 76630094,Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17(Chr17) position 3211643, Chr17 position 30244229, Chr17 position35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18position 19745054, Chr18 position 19747206, Chr18 position 44774403,Chr18 position 55103840, Chr18 position 55106910, Chr18 position70534832, Chr18 position 72880039, Chr18 position 77547934, Chr19position 30016170, Chr19 position 30017283, Chr19 position 30717013,Chr19 position 30719659, Chr20 position 1294019, Chr20 position 3073503,Chr20 position 10198305, Chr20 position 23015989, Chr20 position23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20position 53092916, Chr20 position 59827619, Chr20 position 59828325,Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21position 9826934, or Chromosome 22 position 43807517 with respect tohuman genome assembly hg19.
 45. (canceled)
 46. (canceled)
 47. (canceled)48. (canceled)
 49. (canceled)
 50. (canceled)
 51. (canceled) 52.(canceled)
 53. (canceled)
 54. (canceled)
 55. (canceled)
 56. (canceled)57. The method of claim 3, further comprising: (i) detecting a level ofuracil of said plurality of methylation sites that is equal to orgreater than the level of methylation sites selected from 76.09 for chr1position 11413742, 78.57 for chr1 240934954, 74.49 for chr10 position123778639, 79.31 for chr10 position 123778640, 74.47 for chr11 position1215978, or 83.33 for chr12 position 128750309 with respect to humangenome assembly hg19; and (ii) administering to said subject a treatmentto treat or prevent IDC or directing said subject to obtain treatment totreat or prevent IDC, wherein the treatment comprises (a) lumpectomy,mastectomy, radiation therapy, chemotherapy, or hormone therapy; or (b)administering an active agent comprising trastuzumab, trastuzumabemtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane,anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide,docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus,palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil.58. The method of claim 3, further comprising: (i) detecting a level ofuracil of said plurality of methylation sites that is equal to or lessthan the level of methylation sites selected from 74.32 for chr1position 4714314, 63.81 for chr1 position 39957798, 85.11 for chr146951513, 73.08 for chr1 position 47904912, 71.20 for chr1 position62660691, 46.94 for chr1 position 63785800, 42.86 for chr1 position67600465, 48.28 for chr1 position 91183172, 64.29 for chr1 position166853786, 88.89 for chr1 position 179545096, 89.29 for chr1 position207669851, 82.61 for chr1 position 237205704, 78.57 for chr1 position237205705, 60.00 for chr1 position 240161215, 75.00 for chr10 position8076277, 76.30 for chr10 position 50818610, 50.43 for chr10 position77157527, 67.35 for chr10 position 124902829, 80.00 for chr10 position124909545, 76.74 for chr10 position 130085373, 34.43 for chr10 position134598235, 79.31 for chr10 position 134599080, 65.85 for chr11 position9025912, 51.72 for chr11 position 15963013, 80.95 for chr11 position66187593, 51.52 for chr11 position 71318977, 57.89 for chr11 position101453451, 60.00 for chr12 position 49726711, 69.81 for chr12 position50297756, 82.98 for chr12 position 50297763, 81.13 for chr12 position50297768, 75.47 for chr12 position 50297774, 77.36 for chr12 position50297776, 54.17 for chr12 position 50444766, 52.00 for chr12 position75601447, 36.84 for chr12 position 95941925, 57.14 for chr12 position129338355, 60.71 for chr12 position 129338471, 65.85 for chr13 position28502190, 79.84 for chr13 position 79181509, 70.70 for chr13 position92051154, 73.91 for chr13 position 95363553, 78.95 for chr13 position95363592, 72.36 for chr14 position 29236052, 62.60 for chr14 position29236065, 92.45 for chr14 position 101543886, 69.84 for chr15 position29407958, 73.68 for chr15 position 45403826, 62.30 for chr15 position76630094, 39.68 for chr15 position 89951787, 71.43 for chr17 position35294171, 56.52 for chr17 position 64831307, 43.48 for chr17 position74136562, 62.79 for chr17 position 74865566, 67.01 for chr18 position19745047, 64.10 for chr18 position 19745054, 80.00 for chr18 position19747206, 68.29 for chr18 position 44774403, 66.20 for chr18 position55103840, 50.00 for chr18 position 55106910, 66.67 for chr18 position70534832, 33.33 for chr18 position 77547934, 38.89 for chr19 position30016170, 60.00 for chr19 position 30017283, 54.24 for chr19 position30717013, 66.67 for chr19 position 30719659, 12.20 for chr2 position20870821, 34.25 for chr2 position 45156764, 34.69 for chr2 position74743346, 28.21 for chr2 position 80549703, 63.64 for chr2 position105471544, 64.44 for chr2 position 115919663, 79.31 for chr2 position115920004, 47.06 for chr2 position 118982006, 63.16 for chr2 position177001540, 55.43 for chr20 position 1294019, 62.96 for chr20 position3073503, 62.44 for chr20 position 10198305, 80.70 for chr20 position23015989, 65.59 for chr20 position 23016002, 55.56 for chr20 position26189258, 36.00 for chr20 position 48626669, 72.13 for chr20 position53092916, 58.82 for chr20 position 59827619, 76.47 for chr20 position59828325, 37.32 for chr21 position 9825842, 50.00 for chr21 position9826150, 53.36 for chr21 position 9826934, 40.68 for chr22 position43807517, 57.54 for chr3 position 14852857, 71.96 for chr3 position121903470, 68.63 for chr3 position 170303393, 67.65 for chr3 position170303422, 81.82 for chr3 position 170303423, 56.52 for chr3 position170303424, 69.77 for chr3 position 170303425, 78.38 for chr4 position44449864, 72.00 for chr4 position 54976099, 55.10 for chr4 position56023880, 65.00 for chr5 position 71014951, 80.82 for chr5 position72677229, 55.26 for chr5 position 87981177, 71.83 for chr5 position140743998, 56.25 for chr5 position 178421786, 57.25 for chr6 position41337153, 68.18 for chr6 position 85484102, 55.56 for chr6 position157557787, 75.51 for chr6 position 160769248, 70.45 for chr7 position1282082, 31.03 for chr7 position 32467637, 63.89 for chr7 position71801896, 63.89 for chr7 position 71801905, 55.17 for chr7 position100946148, 43.66 for chr7 position 100946151, 75.00 for chr7 position121957003, 84.83 for chr7 position 150038502, 26.45 for chr7 position157477232, 71.43 for chr7 position 157477399, 92.59 for chr7 position157477401, 70.91 for chr8 position 9764011, 85.51 for chr8 position11566080, 65.22 for chr8 position 11566102, 65.22 for chr8 position11566125, 53.85 for chr8 position 56015232, 95.00 for chr8 position65281933, 44.93 for chr8 position 145105472, 85.19 for chr9 position126780185, 66.96 for chr9 position 127239956, or 84.62 for chr9 position140772369 with respect to human genome assembly hg19; and (ii)administering to said subject a treatment to treat or prevent DC ordirecting said subject to obtain treatment to treat or prevent DC,wherein the treatment comprises (a) lumpectomy, mastectomy, radiationtherapy, chemotherapy, or hormone therapy; or (b) administering anactive agent comprising trastuzumab, trastuzumab emtansine, lapatinib,pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole,doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nabpaclitaxel, eribulin, everolimus, palbociclib, capecitabine,ixabepilone, methotrexate, or fluorouracil.